Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Antibodies to pneumococcal polysaccharides in human milk : lack of relationship to colonization and acute otitis media

BACKGROUND: This study analyzed antibodies to pneumococcal polysaccharides in human milk and their effect on nasopharyngeal colonization and acute otitis media in breast-fed infants.METHODS: A total of 503 milk samples were collected from 310 mothers. Nasopharyngeal cultures were obtained from their children at 2, 6 and 10 months postpartum, and the capsular groups/types of the Streptococcus pneumoniae isolates were determined.RESULTS: Types 6A, 6B, 19A, 19F and 23F accounted for 54% of the pneumococcal isolates, but type 3 isolates were uncommon. Milk samples were analyzed for antibody activity to the common capsular polysaccharide types 6A, 19F and 23F; to the type 3 polysaccharide; to C-polysaccharide; and to phosphorylcholine (PC), a major component of the pneumococcal cell wall polysaccharide (CWPS). Anti-capsular antibody activity was low or absent in > 90% of the milk samples. In contrast anti-PC antibody activity was detected in 88% and anti-CWPS in 84% of the samples. The frequency of acute otitis media did not vary with the milk anti-capsular, anti-PC or anti-CWPS antibody activity.CONCLUSIONS: There was no reduction in nasopharyngeal carriage of S. pneumoniae among children fed milk with anti-capsular or anti-PC antibody activity, but carriage was increased in those children who received milk with anti-CWPS antibody activity. A protective role of antipolysaccharide or anti-CWPS antibodies in milk was not detected under the study conditions

Models for predicting roll roughness progressive loss in cold rolling

Roll roughness retention is one of the critical aspects in cold rolling, since the early loss of roughness in roll surface is responsible of many well known technological problems. Unfortunately, there is a large variety of parameters that may influence such kind of tribosystems and the problem of identifying a reliable prediction model is rather difficult a task. The aim of the present work, developed as part of a larger European Project, namely, TOPROLLS, consists into attempting to achieve a roll roughness loss prediction method that could be suitably used in the plants of interest. The model is restricted to three elementary wear mechanisms: adhesion-abrasion, fatigue, oil trapping effect. The lost volume per unit distance is assumed to be proportional to the dissipated power, and the latter is assumed proportional to the plant rolling power. The wear rate is regarded as a time depending variable, since it depends on the effective contact surface. Fatigue has been taken into account as in a previous investigation. Finally, the influence of surface flattening on lubrication has been considered in order to justify the asymptotic trends of wear and friction evolution with respect to the cycle number. The predicted trends of roughness and friction coefficient are compared to those obtained experimentally in some pilot and factory plants