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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

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level.

The mechanisms by which natural populations of vector mosquitoes cope with daily and seasonal fluctuations in temperature are poorly understood. We examined the effect of water temperature on expression of stress and immune-related genes in Aedes aegypti larvae. Aedes aegypti 3rd instars were exposed for 24 h to one of 7 constant temperatures (10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C [control], 32 degrees C, 36 degrees C, or 40 degrees C) and expression of antimicrobial peptides (cecropin, defensin), transferrin, and heat shock proteins (HSP70 and HSP83) quantified by real-time reverse-transcriptase polymerase chain reaction. Cecropin, defensin, and transferrin were overexpressed at 36 degrees C and underexpressed at 15 degrees C and 32 degrees C. HSP83 was overexpressed at 10 degrees C and 40 degrees C and underexpressed at 20 degrees C, while HSP70 was underexpressed at 15 degrees C, 32 degrees C, and 36 degrees C. These findings suggest that antimicrobial peptides can serve as biomarkers of thermal stress and that HSP83 may buffer mosquito larvae against extreme temperatures.

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.

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.

Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4+T cells from HIV-1-infected donors. Similar experiments were performed with CD4+T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4+T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4+T-cell function.

There is clear evidence that most antimalarial drugs, while acting through different mechanisms, are associated with parasite growth/development inhibition and eventual parasite death. However, the exact mode of parasite death remains unclear. In the present study, we investigated the ability of various drugs, including two antimalarial drugs (chloroquine and atovaquone), a topoisemerase II inhibitor (etoposide) and a nitric oxide donor (S-nitro-N-acetyl-D, L-penicillamine), to induce apoptosis in a laboratory strain of Plasmodium falciparum. Results obtained from flow cytometric analysis showed a significant reduction in the percent of parasitemia and parasite growth in all drug-treated parasite cultures, including those treated with etoposide and S-nitro-N-acetyl-D, L-penicillamine. For further investigation, we used various biochemical approaches including the terminal dUTP nick-end labeling assay, determination of mitochondrial membrane integrity and DNA degradation/fragmentation, to analyze the changes occurring during parasite-drug interactions and eventual death. We observed that loss of membrane potential was induced in parasite cultures treated with atovaquone, while S-nitro-N-acetyl-D, L-penicillamine induced abnormal parasite forms, "crisis forms", and minor DNA degradation. However, these features were not observed in the parasite cultures treated with chloroquine nor were other features of apoptosis-like death associated with any of the drugs used in this study. The death resulting from the various drug treatments is atypical of apotosis. More studies will be needed to define the precise mode of death exhibited by P. falciparum.

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 (

This study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan (pso), a furanocoumarin, on PC-3 and C4-2B castration-resistant prostate cancer (CRPC) cell lines. Pso caused significant G0/G1 cell cycle arrest and inhibition of cell growth. Molecular analysis of cyclin (D1, D2, D3, and E), cyclin-dependent kinase (cdk) (cdks 2, 4, and 6), and cdk inhibitor (p21 and p27) expression suggested transcriptional regulation of the cdk inhibitors and more significant downregulation of cdk4 than of cyclins or other cdks. Overexpression of cdk4, or silencing of p21 or p27, overcame pso-induced G0/G1 arrest, suggesting that G0/G1 cell cycle arrest is a potential mechanism of growth inhibition in CRPC cells.

Malaria infection may be affected by host genetic factors as well as nutritional status. Iron status and the phenotype of haptoglobin, a heme-binding acute phase reactant may be determinants of malaria parasitemia. A combination of cross sectional studies and longitudinal follow-up were used to describe the association between iron status, C-reactive protein, malaria infections and host genetic factors including; haptoglobin (Hp) phenotypes, in children below 9 years in a malaria endemic area in Coastal Kenya. The prevalence of 0.45 and 0.41, respectively for Hp 1-1 and Hp 2-1 phenotypes was significantly higher than 0.14 for Hp 2-2 phenotype (n=162). Children with Hp 2-2 phenotype showed significantly higher iron storage compared to those with Hp 1-1 and Hp 2-1 phenotypes when children with malaria parasites and high C-reactive protein (>9mg/L) were excluded from the analysis. There were no significant differences in malaria parasite densities among Hp phenotypes but children with Hp 2-2 had lower number of clinical malaria episodes (P=0.045). Taken together, this study shows that the presence of malaria may complicate the interpretation of iron status in children based on their Hp-phenotypes. Further studies will be required to address possible interactions among the various genetic factors and iron status in a malaria endemic setting.

HIV-1 infections lead to a progressive depletion of CD4 cells culminating in AIDS. The coreceptor usage by HIV varies from CCR5 (R5) tropic early in infection to CXCR4 (X4) tropic in later infections. Although the coreceptor switch from R5 to X4 tropic HIV is well associated with progression to AIDS, the role of CCR5 in disease progression especially in patients infected exclusively with R5 isolates throughout the disease remains enigmatic. To better understand the role of CCR5 and R5 tropic HIV envelope in AIDS pathogenesis, we asked whether the levels of CCR5 and/or HIV Env-mediated fusion determine apoptosis of bystander cells. We generated CD4(+) T cell lines expressing varying levels of CCR5 on the cell surface to show that CCR5 expression levels correlate with bystander apoptosis induction. The mechanism of apoptosis involved caspase-3 activation and mitochondrial depolarization and was dependent on gp41 fusion activity as confirmed by fusion-restricted gp41 point mutants and use of the fusion inhibitor T20. Interestingly, lower levels of CCR5 were able to support virus replication in the absence of bystander apoptosis. Our findings suggest that R5 HIV-1-mediated bystander apoptosis is dependent on both CCR5 expression levels as well as fusogenic activity of the Env glycoprotein.

Iron deficiency has been reported to affect both malaria pathogenesis and cell-mediated immune responses; however, it is unclear whether the protection afforded by iron deficiency is mediated through direct effects on the parasite, through immune effector functions or through both. We have determined cytokine mRNA expression levels in 59 children living in a malaria endemic area on the coast of Kenya who we selected on the basis of their biochemical iron status. Real-time quantitative reverse transcriptase polymerase chain reaction analysis of cytokine mRNA levels of peripheral blood mononuclear cells (PBMC) obtained from these children showed an association between interleukin-4 (IL-4) mRNA levels and all the biochemical indices of iron that we measured. Furthermore, IL-10 mRNA was higher in parasite blood smear-positive children than in blood smear-negative children irrespective of their iron status. This study suggests that IL-4 expression by PBMC may be affected by iron status.

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.

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 (

Methods To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from eight seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays.

Recapitulating immunodominant A2-restricted HIV-specific CTLs that do not suppress virus in vivo is unlikely to be an effective vaccine strategy. To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from five seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays. One OLP (Gag145-159) was recognized by all donors. Of note, this reactivity predominated in three of five T cell cultures. Fine mapping with progressively truncated peptides revealed three overlapping epitopes: RTLNAWVKV (RV9), RTLNAWVKVV (RV10) and TLNAWVKVV (TV9). TV9 is a known epitope that is rarely recognized in vivo. In contrast, RV9 and RV10 are novel. Although the latter bound with lower affinities to HLA-A2 than TV9, RV9- and RV10-cultures were readily generated. RV9- and RV10-T cells were cytotoxic, capable of secreting many cytokines, and suppressive of HIV replication in vitro. In sum, we report two novel naturally processed and presented epitopes in HIV p24 that are recognized by pre-infection T cell repertoires. Further studies are needed to explain why these reactivities are rare during infections and more importantly, whether this conserved region of the HIV proteome has value as a prophylactic vaccine for A2 individuals.

Background Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to “fever range” hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility. Methods To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed. Results Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells. Conclusion These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock.