Lightweight, open source, easy-use algorithm and web service for paraprotein screening using spatial frequency domain analysis of electrophoresis studies.

Introduction: Serum protein electrophoresis (SPEP) is commonly used to detect monoclonal paraproteins to meet laboratory diagnostic criteria for plasma cell neoplasms. We propose an automated screening method for paraprotein detection that uses minimal computational resources for training and deployment. Methods: A model screening for paraproteins based on the presence of high-frequency components in the spatial frequency spectrum of the SPEP densitometry curve was calibrated on a set of 330 samples, and evaluated on representative (n=110) and external (n=1,321) test sets. The model takes as input a patient's serum densitometry curve and a standardized control curve and outputs a prediction of whether a paraprotein is present. We built an interactive web application allowing users to easily perform paraprotein screening given inputs for densitometry curves, as well as a macro-enabled spreadsheet for easy automated screening. Results: When tuned to maximize likelihood ratio with minimum sensitivity 0.90, the model achieved AUC 0.90, sensitivity 0.90, positive-predictive value 0.64, specificity 0.55, and accuracy 0.72 in the representative test set. In the external test set, the model achieved AUC 0.90, sensitivity 0.97, positive-predictive value 0.42, specificity 0.29, and accuracy 0.52. A subset analysis showed sensitivities of 0.90, 0.96, and 1.0 in detecting low (0.1-0.5 g/dL), medium (0.5-3.0 g/dL), and high paraprotein levels (≥3.0 g/dL), respectively. We have released a web service allowing users to score their own SPEP data, and also released the algorithm and application programming interface in an open-source package allowing users to customize the model to their needs. Conclusions: We developed a proof of concept for an automated method for paraprotein screening using only the characteristics of the SPEP curve. Future work should focus on testing the method with other laboratory data including immunofixation gels, as well as incorporation of outside data sources including clinical data.

Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88(-/-) mice, IL-1R(-/-) mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R(-/-) mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.

Initiation and resolution of mucosal inflammation.

Antigens entering the body through the mucosal surface are screened by a highly developed immune system comprised not only of traditional lymphoid cells but also epithelial cells, fibroblasts, and antigen-presenting cells (APCs). For example, in the intestinal tract, gut-associated lymphoid tissue (GALT) is tolerant to the approx 400 separate commensal strains residing mainly in the colon, but also retains the capacity to detect and remove virulent bacteria before they infect systemically. This review summarizes recent work characterizing the molecular mechanisms involved in acute and chronic intestinal inflammation. We will also describe a natural murine pathogen, Citrobacter rodentium, which is being used to explore the host response to enteric pathogens and the resulting immunopathology.

Protective and destructive innate immune responses to enteropathogenic Escherichia coli and related A/E pathogens.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli (O157:H7) and Citrobacter rodentium are classified as attaching and effacing (A/E) pathogens based on their ability to adhere to intestinal epithelium, destroy microvilli and induce pedestal formation at the site of infection. A/E bacterial infections also cause acute diarrheal episodes and intestinal inflammation. The use of model systems has led to an understanding of the innate immune response to A/E pathogens. The innate immune system plays a protective role, initiating a productive antibody response, directly killing bacteria and inducing repair mechanisms following tissue damage caused by infection. However, hyperactivation of the innate immune system can have negative consequences, including exacerbated tissue destruction following neutrophil infiltration. Here we review how innate immune cell types, including neutrophils, macrophages and dendritic cells, orchestrate both protective and destructive responses. Such information is crucial for the development of therapeutics that can mitigate destructive inflammatory responses while accentuating those that are protective.

TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.

Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene.

Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Moreover we have found that C. elegans genes controlling lifespan (daf-2, age-1 and daf-16), also mediate susceptibility to EPEC. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

IL-4 induces the proteolytic processing of mast cell STAT6.

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.