Automated segmentation of tissue images for computerized IHC analysis

This paper presents two automated methods for the segmentation ofimmunohistochemical tissue images that overcome the limitations of themanual approach aswell as of the existing computerized techniques. The first independent method, based on unsupervised color clustering, recognizes automatically the target cancerous areas in the specimen and disregards the stroma; the second method, based on colors separation and morphological processing, exploits automated segmentation of the nuclear membranes of the cancerous cells. Extensive experimental results on real tissue images demonstrate the accuracy of our techniques compared to manual segmentations; additional experiments show that our techniques are more effective in immunohistochemical images than popular approaches based on supervised learning or active contours. The proposed procedure can be exploited for any applications that require tissues and cells exploration and to perform reliable and standardized measures of the activity of specific proteins involved in multi-factorial genetic pathologie

Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is becoming increasingly complex due to usage of progressively larger panels of reagents and a high number of World Health Organization (WHO) entities. Typically, data analysis is performed separately for each stained aliquot of a sample; subsequently, an expert interprets the overall immunophenotypic profile (IP) of neoplastic B-cells and assigns it to specific diagnostic categories. We constructed a principal component analysis (PCA)-based tool to guide immunophenotypic classification of B-CLPD. Three reference groups of immunophenotypic data files—B-cell chronic lymphocytic leukemias (B-CLL; n=10), mantle cell (MCL; n=10) and follicular lymphomas (FL; n=10)—were built. Subsequently, each of the 175 cases studied was evaluated and assigned to either one of the three reference groups or to none of them (other B-CLPD). Most cases (89%) were correctly assigned to their corresponding WHO diagnostic group with overall positive and negative predictive values of 89 and 96%, respectively. The efficiency of the PCA-based approach was particularly high among typical B-CLL, MCL and FL vs other B-CLPD cases. In summary, PCA-guided immunophenotypic classification of B-CLPD is a promising tool for standardized interpretation of tumor IP, their classification into well-defined entities and comprehensive evaluation of antibody panels

Reassessing the role of DotF in the Legionella pneumophila type IV secretion system.

Legionella pneumophila, the causative agent of a severe pneumonia termed Legionnaires' Disease, survives and replicates within both protozoan hosts and human alveolar macrophages. Intracellular survival is dependent upon secretion of a plethora of protein effectors that function to form a replicative vacuole, evade the endocytic pathway and subvert host immune defenses. Export of these factors requires a type IV secretion system (T4SS) called Dot/Icm that is composed of twenty-seven proteins. This report focuses on the DotF protein, which was previously postulated to have several different functions, one of which centered on binding Dot/Icm substrates. In this report, we examined if DotF functions as the T4SS inner membrane receptor for Dot/Icm substrates. Although we were able to recapitulate the previously published bacterial two-hybrid interaction between DotF and several substrates, the interaction was not dependent on the Dot/Icm substrates' signal sequences as predicted for a substrate:receptor interaction. In addition, binding did not require the cytoplasmic domain of DotF, which was anticipated to be involved in recognizing substrates in the cytoplasm. Finally, inactivation of dotF did not abolish intracellular growth of L. pneumophila or translocation of substrates, two phenotypes dependent on the T4SS receptor. These data strongly suggest that DotF does not act as the major receptor for Dot/Icm substrates and therefore likely performs an accessory function within the core-transmembrane subcomplex of the L. pneumophila Dot/Icm type IV secretion system

Interaction between DotF and substrates is not mediated by the secretion signal sequence.

<p>(A) Visualization of the two-hybrid interaction on MacConkey indicator media. Shown is a portion of a plate containing a red positive control consisting of two leucine zippers fused to the T18 and T25 domains of CyaA (1) and a white negative control consisting of the T18 and T25 empty vector controls (2). The T18:DotF(29–123) fragment was assayed for interaction with T25:RalF and T25:SidG containing their signal sequences (3–4) versus fusions lacking their signal sequences (6–7) and the T25 vector control (5 and 8). (B) Quantification of the interactions in (A) by measurement of β-galactosidase activity. (C,D) The secretion signal sequence is required for translocation. CyaA:RalF (black bars), CyaA:RalF(ΔSS) (white bars), CyaA:SidG (black bars), CyaA:SidG(ΔSS) (white bars) were assayed for secretion in a wild type strain (WT) or a <i>dotA</i> mutant strain that is defective for secretion (T4SS⁻). Secretion was monitored by an ELISA assay that detects calmodulin-induced cAMP production. Assays were performed in triplicate and error bars represent the standard deviation from the mean.</p

Schematic of the <i>L.</i><i>pneumophila</i> type IV secretion system (T4SS).

<p>The 27 proteins of the T4SS are shown at their predicted or experimentally determined subcellular locations in the outer membrane (OM), periplasm, inner membrane (IM) and cytoplasm. Dot proteins are labeled with the last letter of their name. Icm proteins are designated in the same manner, but are prefaced with an ‘i’.</p