Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

Effect of furosemide on body composition and urinary proteins that mediate tubular sodium and sodium transport-A randomized controlled trial.

BACKGROUND: Furosemide inhibits the sodium potassium chloride cotransporter (NKCC2) in the thick ascending limb of the loop of Henle and increases urinary water and sodium excretion. This study investigates the effect of furosemide on body composition estimated with multifrequency bioimpedance spectroscopy (BIS) technique and urinary proteins from NKCC2. METHODS: This study is a randomized, placebo-controlled, crossover study where healthy subjects received either placebo or 40 mg furosemide on two separate occasions, where body composition with BIS, renal function, proteins from tubular proteins that mediate sodium and water transport, and plasma concentrations of vasoactive hormones were measured before and after intervention. RESULTS: We observed an expected increased diuresis with a subsequent reduction in bodyweight of (-1.51 ± 0.36 kg, p < .001) and extracellular water (ECW; -1.14 ± 0.23 L, p < .001) after furosemide. We found a positive correlation between the decrease in ECW and a decrease in bodyweight and a negative correlation between the decrease in ECW and the increase in urinary output. Intracellular water (ICW) increased (0.47 ± 0.28 L, p < .001). Urinary excretion of NKCC2 increased after furosemide and the increase in NKCC2 correlated with an increase in urine output and a decrease in ECW. CONCLUSION: We found BIS can detect acute changes in body water content but the method may be limited to estimation of ECW. BIS demonstrated that furosemide increases ICW which might be explained by an extracellular sodium loss. Finally, urinary proteins from NKCC2 increases after furosemide with a good correlation with diuresis end the decrease in ECW.

Genetic disruption of slc4a10 alters the capacity for cellular metabolism and vectorial ion transport in the choroid plexus epithelium.

BACKGROUND: Genetic disruption of slc4a10, which encodes the sodium-dependent chloride/bicarbonate exchanger Ncbe, leads to a major decrease in Na+-dependent HCO3- import into choroid plexus epithelial cells in mice and to a marked reduction in brain intraventricular fluid volume. This suggests that Ncbe functionally is a key element in vectorial Na+ transport and thereby for cerebrospinal fluid secretion in the choroid plexus. However, slc4a10 disruption results in severe changes in expression of Na+,K+-ATPase complexes and other major transport proteins, indicating that profound cellular changes accompany the genetic manipulation. METHODS: A tandem mass tag labeling strategy was chosen for quantitative mass spectrometry. Alterations in the broader patterns of protein expression in the choroid plexus in response to genetic disruption of Ncbe was validated by semi-quantitative immunoblotting, immunohistochemistry and morphometry. RESULTS: The abundance of 601 proteins were found significantly altered in the choroid plexus from Ncbe ko mice relative to Ncbe wt. In addition to a variety of transport proteins, particularly large changes in the abundance of proteins involved in cellular energy metabolism were detected in the Ncbe ko mice. In general, the abundance of rate limiting glycolytic enzymes and several mitochondrial enzymes were reduced following slc4a10 disruption. Surprisingly, this was accompanied by increased ATP levels in choroid plexus cells, indicating that the reduction in capacity for energy metabolism was adaptive to high ATP rather than causal for a decreased capacity for ion and water transport. Ncbe-deficient cells also had a reduced cell area and decreased K+ content. CONCLUSION: Our findings suggest that the lack of effective Na+-entry into the epithelial cells of the choroid plexus leads to a profound change in the cellular phenotype, shifting from a high-rate secretory function towards a more dormant state; similar to what is observed during ageing or Alzheimer's disease.

The TRPV5 promoter as a tool for generation of transgenic mouse models.

The transient receptor potential vanilloid 5 (TRPV5) is a Ca(2+) channel, which is expressed in renal late distal convoluted tubules (DCT2s) and connecting tubules (CNTs). These tubules play a major role in hormone controlled renal Ca(2+) reabsorption, and thereby in body Ca(2+) homeostasis, as well as urinary excretion of other electrolytes, including Na(+) and K(+). DCT2 and CNT are difficult to distinguish from the surrounding structures and thereby to study by direct functional methods. We developed a transgenic mouse model expressing enhanced green fluorescent protein (EGFP) driven by the TRPV5 promoter to identify these specific tubules. Expression of EGFP in the DCT2 and CNT allows the isolation of pure DCT2 and CNT populations for proteomic and physiological analyses. The TRPV5 promoter is also useful for generating conditional knockout mouse models in a cell-specific manner. TRPV5 promoter driven Cre recombinase expression will be useful for inducing DCT2 and CNT specific gene silencing of various channels, pumps, carriers, and receptors. In this chapter, we describe the strategy for developing transgenic mouse lines involving the TRPV5 promoter, provide a description of extensive validation of these mouse lines, and discuss possible uses and limitations.