Defining clinical malaria: the specificity and incidence of endpoints from active and passive surveillance of children in rural Kenya

Background

Febrile malaria is the most common clinical manifestation of P. falciparum infection, and is often the primary endpoint in clinical trials and epidemiological studies. Subjective and objective fevers are both used to define the endpoint, but have not been carefully compared, and the relative incidence of clinical malaria by active and passive case detection is unknown.

Methods

We analyzed data from cohorts under active and passive surveillance, including 19,462 presentations with fever and 5,551 blood tests for asymptomatic parasitaemia. A logistic regression model was used to calculate Malaria Attributable Fractions (MAFs) for various case definitions. Incidences of febrile malaria by active and passive surveillance were compared in a subset of children matched for age and location.

Results

Active surveillance identified three times the incidence of clinical malaria as passive surveillance in a subset of children matched for age and location. Objective fever (temperature≥37.5°C) gave consistently higher MAFs than case definitions based on subjective fever.

Conclusion

The endpoints from active and passive surveillance have high specificity, but the incidence of endpoints is lower on passive surveillance. Subjective fever had low specificity and should not be used in primary endpoint. Passive surveillance will reduce the power of clinical trials but may cost-effectively deliver acceptable sensitivity in studies of large populations.     

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.     

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.     

Profiles of human serum antibody responses elicited by three leading HIV vaccines focusing on the induction of Env-specific antibodies

In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN) Study #041 (recombinant protein alone), HVTN Study #203 (poxviral vector prime-protein boost), and the DP6-001 study (DNA prime-protein boost). Vaccinees from HVTN Study #041 had the highest neutralizing antibody activities against the sensitive virus along with the highest binding antibody responses, particularly those directed toward the V3 loop. DP6-001 sera showed a higher frequency of positive neutralizing antibody activities against more resistant viral isolate with a significantly higher CD4 binding site (CD4bs) antibody response compared to both HVTN studies #041 and #203. No differences were found in CD4-induced (CD4i) antibody responses, ADCC activity, or complement activation by Env-specific antibody among these sera. Given recent renewed interest in realizing the importance of antibody responses for next generation HIV vaccine development, different antibody profiles shown in the current report, based on the analysis of a wide range of antibody parameters, provide critical biomarker information for the selection of HIV vaccines for more advanced human studies and, in particular, those that can elicit antibodies targeting conformational-sensitive and functionally conserved epitopes.     

Equivalence of ELISpot assays demonstrated between major HIV network laboratories

Background

The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.

Methods

We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.

Findings

Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.

Interpretation

The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.     

The Effect of Opioid Receptor Blockade on the Neural Processing of Thermal Stimuli

The endogenous opioid system represents one of the principal systems in the modulation of pain. This has been demonstrated in studies of placebo analgesia and stress-induced analgesia, where anti-nociceptive activity triggered by pain itself or by cognitive states is blocked by opioid antagonists. The aim of this study was to characterize the effect of opioid receptor blockade on the physiological processing of painful thermal stimulation in the absence of cognitive manipulation. We therefore measured BOLD (blood oxygen level dependent) signal responses and intensity ratings to non-painful and painful thermal stimuli in a double-blind, cross-over design using the opioid receptor antagonist naloxone. On the behavioral level, we observed an increase in intensity ratings under naloxone due mainly to a difference in the non-painful stimuli. On the neural level, painful thermal stimulation was associated with a negative BOLD signal within the pregenual anterior cingulate cortex, and this deactivation was abolished by naloxone.     

Effects of dietary supplementation of DL-methionine or 2-hydroxy-4-(methylthio)-butanoic acid on food intake, nutrient digestibility, nitrogen balance, and urinary and blood metabolites in healthy, growing dogs

The aim of this study was to evaluate effects on nutritional responses of supplemental DL-methionine and 2-hydroxy-4-(methylthio) butanoic acid (HMTBA) in a commercial-type diet in growing dogs. A nitrogen balance study was conducted as a randomized complete block design using 30 Pointer puppies (72-d-old; 5.5 kg). A corn and poultry byproduct meal based diet was supplemented with 0.1 or 0.2% DL-methionine or HMTBA on an equimolar basis. Organic matter and gross energy tended (p < 0.10) to be less digestible by dogs fed the 0.1% HMTBA diet compared with the 0.2% DL-methionine diet, but other nutrients were unaffected. Postprandial urinary calcium tended (p < 0.10) to be lower for the basal and HMTBA treatments. Fecal ammonia tended (p < 0.10) to be lower for the 0.1% HMTBA diet than for the 0.2% DL-methionine diet. At the levels tested, DL-methionine and HMTBA appear to act similarly when included in a corn and poultry by-product meal diet fed to young dogs.     

Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography

Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Antioxidant properties of the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): a defense against environmental pro-oxidants?

Polychaeta species like Laeonereis acuta (Nereididae) usually secrete great amounts of mucus that wrap the animal inside. Taking into account that fungi action in the sediment and UV radiation acting on dissolved organic matter in the water produces reactive oxygen species (ROS) like hydrogen peroxide (H(2)O(2)), it was considered that the mucus secretion could represent an antioxidant defense against environmental ROS. Antioxidant enzymes (catalase-CAT; superoxide dismutase-SOD; glutathione peroxidase-GPx and glutathione-S-transferase-GST) and total antioxidant capacity (TOSC) were determined in worms and mucus secretion. Higher (p<0.05) CAT, GPx and TOSC values were registered in mucus samples respect worms, SOD activity was similar (p>0.05) in both kind of samples, and absence of GST activity was observed in mucus samples, suggesting absence of catalyzed phase II reactions. In assays conducted with hepatoma cell lines exposed to H(2)O(2), it was verified that: (1) mucus co-exposure significantly (p<0.05) lowered DNA damage induced by H(2)O(2); (2) ROS production was significantly (p<0.05) reduced when cells were exposed simultaneously with mucus samples and H(2)O(2) respect H(2)O(2) alone. It can be concluded that the mucus production contributes substantially to the antioxidant defense system of the worm against environmental ROS through the interception or degradation of H(2)O(2), peroxyl and hydroxyl radicals.