James E. Kirby

The Alzheimer's Disease-Associated Amyloid β-Protein Is an Antimicrobial Peptide

Background The amyloid β-protein (Aβ) is believed to be the key mediator of Alzheimers disease (AD) pathology. Aβ is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Aβ has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities. Methodology/Principal Findings Here, we provide data supporting an in vivo function for Aβ as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Aβ and LL-37, an archetypical human AMP. Findings reveal that Aβ exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Aβ levels. Consistent with Aβ-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Aβ antibodies. Conclusions/Significance Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies.

Xist RNA Is a Potent Suppressor of Hematologic Cancer in Mice

X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.

Anthrax Lethal Toxin Induces Human Endothelial Cell Apoptosis

ABSTRACT Because of its ease of dispersal and high lethality, Bacillus anthracis is one of the most feared biowarfare agents. A better understanding of anthrax pathogenesis is urgently needed to develop new therapies for systemic disease that is relatively unresponsive to antibiotics. Although experimental evidence has implicated a role for macrophages in anthrax pathogenesis, clinical and pathological observations suggest that a direct insult to the host vasculature may also be important. Two bacterial toxins, lethal toxin and edema toxin, are believed to mediate the clinical sequelae of anthrax. Here, I examined whether these toxins are directly toxic to endothelial cells, the cell type that lines the interior of blood vessels. I show for the first time that lethal toxin but not edema toxin reduces the viability of cultured human endothelial cells and induces caspase-dependent endothelial apoptosis. In addition, this toxicity affects both microvascular and large vessel endothelial cells as well as endothelial cells that have differentiated into tubules within a type I collagen extracellular matrix. Finally, lethal toxin induces cleavage of mitogen-activated protein kinase kinases in endothelial cells and inhibits phosphorylation of ERK, p38, and JNK p46. Based on the contributions of these pathways to endothelial survival, I propose that lethal toxin-mediated cytotoxicity/apoptosis results primarily through inhibition of the ERK pathway. I also hypothesize that the observed endothelial toxicity contributes to vascular pathology and hemorrhage during systemic anthrax.

Bartonella-associated endothelial proliferation depends on inhibition of apoptosis

Bartonella is a Gram-negative pathogen that is unique among bacteria in being able to induce angioproliferative lesions. Cultured human endothelial cells have provided an in vitro system in which to study the basis of angioproliferation. Previous studies have attributed the organisms ability to induce angioproliferative lesions to direct mitotic stimulation of endothelial cells by these bacteria. Here we show that Bartonella inhibits apoptosis of endothelial cells in vitro, and that its ability to stimulate proliferation of endothelial cells depends to a large extent on its antiapoptotic activity. Bartonella suppresses both early and late events in apoptosis, namely caspase activation and DNA fragmentation, respectively. Its ability to inhibit death of endothelial cells after serum starvation can be recapitulated by media conditioned by bacteria, indicating that direct cell contact is not necessary. Among tested strains, the activity is produced only by Bartonella species that are significant human pathogens and are associated with angioproliferative lesions. We suggest that endothelial cells normally respond to infection by undergoing apoptosis and that Bartonella evolved the antiapoptotic activity to enhance survival of the host cells and therefore itself. We propose that Bartonellas antiapoptotic mechanism accounts at least in part for its ability to induce vascular proliferation in vivo.

Ruling out novel H1N1 influenza virus infection with direct fluorescent antigen testing.

We evaluated the ability of direct fluorescent antigen (DFA) influenza tests to identify novel H1N1 influenza virus. DFA results were compared with polymerase chain reaction results. The negative predictive value of DFA testing was at least 96%. Therefore, when performed on specimens of adequate quality, DFA tests can effectively rule out infection due to novel H1N1 virus.

Evaluation of the Abbott RealTime™ CT/NG assay in comparison to the Roche COBAS AMPLICOR CT/NG assay

ABSTRACT Several commercial methods exist for the molecular detection of Chlamydia trachomatis and Neisseria gonorrhoeae in clinical samples. Here we evaluated the performance characteristics of the newly FDA-cleared Abbott RealTime CT/NG assay (where “CT” stands for Chlamydia trachomatis and “NG” stands for Neisseria gonorrhoeae) that uses the automated m2000 molecular platform. Results were compared to those of the Roche Cobas Amplicor CT/NG assay. A total of 926 cervical swab, 45 female urine, 6 male urethral swab, and 407 male urine specimens from 1,384 patients were examined. After resolving all Roche N. gonorrhoeae-positive results with two additional real-time PCR assays, we found that the agreement between the assays was excellent. For urine samples, there was 99.6% positive agreement and 97.7% negative agreement for C. trachomatis, and for male urine samples, there was 100% positive agreement and 99.7% negative agreement for N. gonorrhoeae. For cervical swab samples, there was 98.8% positive agreement and 98.5% negative agreement for C. trachomatis, and there was 96.6% positive agreement and 99.8% negative agreement for N. gonorrhoeae. In limiting dilution analyses, we found that the Abbot assay was more sensitive than the Roche assay for both C. trachomatis and N. gonorrhoeae. In addition, there appeared to be an enhanced ability of the Abbott assay to detect dual infections, especially in the presence of large amounts of N. gonorrhoeae and small amounts of C. trachomatis organisms. In summary, we conclude that the Abbott RealTime CT/NG assay is an accurate and automated new addition to the available testing options for C. trachomatis and N. gonorrhoeae.

Rapid Identification of Staphylococcus aureus in Blood Cultures by Use of the Direct Tube Coagulase Test

ABSTRACT Direct tube coagulase testing for identification of Staphylococcus aureus from BACTEC culture broth showed a sensitivity, a specificity, and positive and negative predicative values of 34%, 100%, 100%, and 80.2% with 2 h of incubation and 65%, 98.7, 99.7%, and 88.6% with 4 h of incubation. Anaerobic blood culture contributed significantly to the detection of S. aureus.

In vitro model of Bartonella henselae-induced angiogenesis.

ABSTRACT Bartonella henselae is a gram-negative pathogen that causes angiogenesis. Here, I establish in vitro models to study Bartonella-induced blood vessel formation. I found that B. henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks

Significance Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat: as an increasing cause of disease and as the staging ground for resistance to “last line” drugs. Thus, we must understand how CRE evolve, diversify, and spread and especially the potential for asymptomatic transmission without outbreaks. Our broad sample of species and genetic determinants that defined four hospital CRE communities over 16 mo revealed a significant degree of CRE diversity, with little evidence for clonal spread but extensive movement of resistance determinants. We provide evidence for considerable asymptomatic carriage and unrecognizable mechanisms of carbapenem resistance that, together, indicate continued innovation by these organisms to thwart the action of this important class of antibiotics and underscore the need for continued surveillance of CRE. Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae. There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Histopathology in a murine model of anthrax

Systemic anthrax infection is usually fatal even with optimal medical care. Further insights into anthrax pathogenesis are therefore urgently needed to develop more effective therapies. Animal models that reproduce human disease will facilitate this research. Here, we describe the detailed histopathology of systemic anthrax infection in A/J mice infected with Bacillus anthracis Sterne, a strain with reduced virulence for humans. Subcutaneous infection leads to systemic disease with multiple pathologies including oedema, haemorrhage, secondary pneumonia and lymphocytolysis. These pathologies bear marked similarity to primary pathologies observed during human disease. Therefore, this simple, small animal model will allow researchers to study the major pathologies observed in humans, while permitting experimentation in more widely available Biosafety Level 2 facilities.