Automated Discrimination of Pathological Regions in Tissue Images: Unsupervised Clustering vs Supervised SVM Classification

Recognizing and isolating cancerous cells from non pathological tissue areas (e.g. connective stroma) is crucial for fast and objective immunohistochemical analysis of tissue images. This operation allows the further application of fully-automated techniques for quantitative evaluation of protein activity, since it avoids the necessity of a preventive manual selection of the representative pathological areas in the image, as well as of taking pictures only in the pure-cancerous portions of the tissue. In this paper we present a fully-automated method based on unsupervised clustering that performs tissue segmentations highly comparable with those provided by a skilled operator, achieving on average an accuracy of 90%. Experimental results on a heterogeneous dataset of immunohistochemical lung cancer tissue images demonstrate that our proposed unsupervised approach overcomes the accuracy of a theoretically superior supervised method such as Support Vector Machine (SVM) by 8%

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

HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4.

For efficient entry into target cells, certain T cell-tropic HIV-1 isolates require both CD4 and the coreceptor CXCR4. However, the molecular interactions among CD4, CXCR4, and the HIV-1 envelope glycoproteins are only now being elucidated. Here we show that the binding of soluble gp120 from one macrophage-tropic and four T cell-tropic viruses to a CD4+, but not to a CD4-, T cell line, decreased the binding of an mAb specific for CXCR4 to its epitope, implying an interaction among gp120, CD4, and CXCR4. To confirm such an interaction, we conducted double- and triple-color confocal laser scanning microscopy on CD4+/CXCR4+ cells and determined the extent of CD4 and CXCR4 colocalization by a semiquantitative analysis. In the absence of gp120, a low level of constitutive colocalization between CD4 and CXCR4 was observed. Treatment with T cell-tropic-derived gp120 and, to a lesser extent, macrophage-tropic-derived gp120, increased the colocalization of CD4 with CXCR4, and triple staining indicated that gp120 was associated with the CD4-CXCR4 complexes. Cocapping of the gp120-CD4-CXCR4 complexes at 37 degrees C resulted in the cointernalization of a proportion of the gp120-CXCR4 complexes into intracellular vesicles. These data demonstrate that the binding of gp120 to CD4+ T cells induces the formation of a trimolecular complex consisting of gp120, CD4, and the HIV-1 coreceptor molecule CXCR4