IL-12Rβ2 is essential for the development of experimental cerebral malaria

A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rβ2 in ECM development. C57BL/6 mice deficient for IL-12Rβ2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rβ2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rβ2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rβ2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rβ2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rβ2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.     

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.     

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease.     

Cyclopiazonic acid-induced changes in the contraction and Ca2+ transient of frog fast-twitch skeletal muscle

The effects ofcyclopiazonic acid (CPA) were investigated on isolated skeletal musclefibers of frog semitendinosus muscle. CPA (0.5-10 µM) enhancedisometric twitch but produced little change in resting tension. Athigher concentrations (10-50 µM), CPA depressed twitch andinduced sustained contracture without affecting resting and actionpotentials. In Triton-skinned fibers, CPA had no significant effect onmyofibrillar Ca²⁺ sensitivity butdecreased maximal activated force at concentrations >5 µM. Inintact cells loaded with the Ca²⁺fluorescence indicator indo 1, CPA (2 µM) induced an increase inCa²⁺-transient amplitude (10 ± 2.5%), which was associated with an increase in time to peak and inthe time constant of decay. Consequently, peak force was increased by35 ± 4%, and both time to peak and the time constant of relaxationwere prolonged. It is concluded that CPA effects, at a concentration ofup to 2 µM, were associated with specific inhibition of sarcoplasmicreticulumCa²⁺-adenosinetriphosphatase inintact skeletal muscle and that inhibition of the pump directlyaffected the handling of intracellularCa²⁺ and force production.

     

Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms

Background

We conducted a tuberculosis (TB) prevalence survey and evaluated the screening methods used in our survey, to assess if screening in TB prevalence surveys could be simplified, and to assess the accuracy of screening algorithms that may be applicable for active case finding.

Methods

All participants with a positive screen on either a symptom questionnaire, chest radiography (CXR) and/or sputum smear microscopy submitted sputum for culture. HIV status was obtained from prevalent cases. We estimated the accuracy of modified screening strategies with bacteriologically confirmed TB as the gold standard, and compared these with other survey reports. We also assessed whether sequential rather than parallel application of symptom, CXR and HIV screening would substantially reduce the number of participants requiring CXR and/or sputum culture.

Results

Presence of any abnormality on CXR had 94% (95%CI 88–98) sensitivity (92% in HIV-infected and 100% in HIV-uninfected) and 73% (95%CI 68–77) specificity. Symptom screening combinations had significantly lower sensitivity than CXR except for ‘any TB symptom’ which had 90% (95%CI 84–95) sensitivity (96% in HIV-infected and 82% in HIV-uninfected) and 32% (95%CI 30–34) specificity. Smear microscopy did not yield additional suspects, thus the combined symptom/CXR screen applied in the survey had 100% (95%CI 97–100) sensitivity. Specificity was 65% (95%CI 61–68). Sequential application of first a symptom screen for ‘any symptom’, followed by CXR-evaluation and different suspect criteria depending on HIV status would result in the largest reduction of the need for CXR and sputum culture, approximately 36%, but would underestimate prevalence by 11%.

Conclusion

CXR screening alone had higher accuracy compared to symptom screening alone. Combined CXR and symptom screening had the highest sensitivity and remains important for suspect identification in TB prevalence surveys in settings where bacteriological sputum examination of all participants is not feasible.