Relative Validity of Three Food Frequency Questionnaires for Assessing Dietary Intakes of Guatemalan Schoolchildren

ObjectiveTo determine the relative validity of three food frequency questionnaires (FFQs) compared with results from 24-hour dietary recalls for measuring dietary intakes in Guatemalan schoolchildren.DesignA cross-sectional study of primary caregivers (mothers or grandmothers) of 6–11 year-old children. Caregivers completed one of three constructed FFQs to measure the child’s dietary consumption in the last week: FFQ1 did not incorporate portion sizes; FFQ2 provided portion sizes; and FFQ3 incorporated pictures of median portion sizes. During the same week, each caregiver also completed three 24-hour dietary recalls. Results from the FFQ were compared with corresponding results from the 24-hour dietary recalls.SettingSanta Catarina Pinula, peri-urban Guatemala City.SubjectsCaregivers (n = 145) of 6–11 year-old children: 46 completed FFQ1, 49 completed FFQ2, and 50 completed FFQ3.ResultsThe mean values for all nutrients obtained from the 24-hour dietary recall were lower than for those obtained from the FFQs, excluding folic acid in FFQ3, cholesterol and zinc in FFQ2, and cholesterol, folic acid, magnesium, potassium, sodium, and zinc in FFQ1. Energy-adjusted Pearson correlation coefficients ranged from 0.07 (protein) to 0.54 (cholesterol) for FFQ1 and from 0.05 to 0.74 for FFQ2 and FFQ3. Agreement by both methods (FFQ and 24-hour dietary recalls) of classifying children into the same or adjacent quartiles of energy-adjusted nutrient consumption ranged from 62.0% for cholesterol to 95.9% for vitamin B12 across all three FFQs.ConclusionsOur FFQs had moderate to good relative validity in measuring energy and nutrient intakes for 6–11 year-old Guatemalan children. More evidence is needed to evaluate their reproducibility and applicability in similar populations.     

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca²⁺ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.     

KAP1 Deacetylation by SIRT1 Promotes Non-Homologous End-Joining Repair

Homologous recombination and non-homologous end joining are two major DNA double-strand-break repair pathways. While HR-mediated repair requires a homologous sequence as the guiding template to restore the damage site precisely, NHEJ-mediated repair ligates the DNA lesion directly and increases the risk of losing nucleotides. Therefore, how a cell regulates the balance between HR and NHEJ has become an important issue for maintaining genomic integrity over time. Here we report that SIRT1-dependent KAP1 deacetylation positively regulates NHEJ. We show that up-regulation of KAP1 attenuates HR efficiency while promoting NHEJ repair. Moreover, SIRT1-mediated KAP1 deacetylation further enhances the effect of NHEJ by stabilizing its interaction with 53BP1, which leads to increased 53BP1 focus formation in response to DNA damage. Taken together, our study suggests a SIRT1-KAP1 regulatory mechanism for HR-NHEJ repair pathway choice.     

Predicting Stroke Risk Based on Health Behaviours: Development of the Stroke Population Risk Tool (SPoRT)

Background

Health behaviours, important factors in cardiovascular disease, are increasingly a focus of prevention. We appraised whether stroke risk can be accurately assessed using self-reported information focused on health behaviours.

Methods

Behavioural, sociodemographic and other risk factors were assessed in a population-based survey of 82 259 Ontarians who were followed for a median of 8.6 years (688 000 person-years follow-up) starting in 2001. Predictive algorithms for 5-year incident stroke resulting in hospitalization were created and then validated in a similar 2007 survey of 28 605 respondents (median 4.2 years follow-up).

Results

We observed 3 236 incident stroke events (1 551 resulting in hospitalization; 1 685 in the community setting without hospital admission). The final algorithms were discriminating (C-stat: 0.85, men; 0.87, women) and well-calibrated (in 65 of 67 subgroups for men; 61 of 65 for women). An index was developed to summarize cumulative relative risk of incident stroke from health behaviours and stress. For men, each point on the index corresponded to a 12% relative risk increase (180% risk difference, lowest (0) to highest (9) scores). For women, each point corresponded to a 14% relative risk increase (340% difference, lowest (0) to highest (11) scores). Algorithms for secondary stroke outcomes (stroke resulting in death; classified as ischemic; excluding transient ischemic attack; and in the community setting) had similar health behaviour risk hazards.

Conclusion

Incident stroke can be accurately predicted using self-reported information focused on health behaviours. Risk assessment can be performed with population health surveys to support population health planning or outside of clinical settings to support patient-focused prevention.     

Phenotypic and Functional Characterization of Monoclonal Antibodies with Specificity for Rhesus Macaque CD200, CD200R and Mincle

Lectin-like molecules and their receptors are cell surface molecules that have been shown to play a role in either facilitating infection or serving as transporters of HIV/SIV in vivo. The role of these lectin-like molecules in the pathogenesis of HIV/SIV infection continues to be defined. In efforts to gain further insight on the potential role of these lectin-like molecules, our laboratory generated monoclonal antibodies (mAb) against the human analogs of rhesus macaque CD200, CD200R and Mincle, since the rhesus macaques are accepted as the most reliable animal model to study human HIV infection. The characterization of the cell lineages from the blood and various tissues of rhesus macaques that express these lectin-like molecules are described herein. Among the mononuclear cells, the cells of the myeloid lineage of rhesus macaques are the predominant cell lineages that express readily detectable levels of CD200, CD200R and Mincle that is similar to the expression of Siglec-1 and Siglec-3 reported by our laboratory earlier. Subset analysis revealed that a higher frequency of the CD14⁺/CD16^- subset from normal rhesus macaques express CD200, CD200R and Mincle. Differences in the frequencies and density of expression of these molecules by the gated population of CD14⁺ cells from various tissues are noted with PBMC and bone marrow expressing the highest and the mononuclear cells isolated from the colon and ileum expressing the lowest levels. While a significant frequency of pDCs and mDCs express Siglec-1/Siglec-3, a much lower frequency expresses CD200, CD200R and Mincle in PBMCs from rhesus macaques. The mAb against CD200 and CD200R but not Mincle appear to inhibit the infection of macrophage tropic SIV/SHIV in vitro. We conclude that these mAbs may have potential to be used as adjunctive therapeutic agents to control/inhibit SIV/HIV infection.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Interleukin (IL)-6 and IL-10 Are Up Regulated in Late Stage Trypanosoma brucei rhodesiense Sleeping Sickness

BackgroundSleeping sickness due to Trypanosoma brucei rhodesiense has a wide spectrum of clinical presentations coupled with differences in disease progression and severity across East and Southern Africa. The disease progresses from an early (hemo-lymphatic) stage to the late (meningoencephalitic) stage characterized by presence of parasites in the central nervous system. We hypothesized that disease progression and severity of the neurological response is modulated by cytokines.MethodsA total of 55 sleeping sickness cases and 41 healthy controls were recruited passively at Lwala hospital, in Northern Uganda. A panel of six cytokines (IFN-γ, IL1-β, TNF-α, IL-6, TGF-β and IL-10) were assayed from paired plasma and cerebrospinal fluid (CSF) samples. Cytokine concentrations were analyzed in relation to disease progression, clinical presentation and severity of neurological responses.ResultsMedian plasma levels (pg/ml) of IFN-γ (46.3), IL-6 (61.7), TGF-β (8755) and IL-10 (256.6) were significantly higher in cases compared to controls (p< 0.0001). When early stage and late stage CSF cytokines were compared, IL-10 and IL-6 were up regulated in late stage patients and were associated with a reduction in tremors and cranioneuropathy. IL-10 had a higher staging accuracy with a sensitivity of 85.7% (95% CI, 63.7%-97%) and a specificity of 100% (95% CI, 39.8%-100%) while for IL-6, a specificity of 100% (95% CI, 47.8%-100%) gave a sensitivity of 83.3% (95% CI, 62.2%-95.3%).ConclusionOur study demonstrates the role of host inflammatory cytokines in modulating the progression and severity of neurological responses in sleeping sickness. We demonstrate here an up-regulation of IL-6 and IL-10 during the late stage with a potential as adjunct stage biomarkers. Given that both cytokines could potentially be elevated by other CNS infections, our findings should be further validated in a large cohort of patients including those with other inflammatory diseases such as cerebral malaria.     

Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.     

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer’s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer’s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer’s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.