Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii.

Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii Potential orthologs of Pca1 have been identified in P. jirovecii Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

Cellular and subcellular alternans in the canine left ventricle.

Previous studies indicate that action potential duration (APD) alternans is initiated in the endocardial (END) and midmyocardial (MID) regions rather than the epicardium (EPI) in the canine left ventricle (LV). This study examines regional differences in the rate dependence of Ca(2+) transient characteristics under conditions that give rise to APD and associated T wave alternans. The role of the sarcoplasmic reticulum (SR) was further evaluated by studying Ca(2+) transient characteristics in myocytes isolated from neonates, where an organized SR is poorly developed. All studies were performed in cells and tissues isolated from the canine LV. Isolated canine ENDO, MID, and EPI LV myocytes were either field stimulated or voltage clamped, and Ca(2+) transients were measured by confocal microscopy. In LV wedge preparations, increasing the basic cycle length (BCL) from 800 to 250 ms caused alternans to appear mainly in the ENDO and MID region; alternans were not observed in EPI under these conditions. Ca(2+) transient alternans developed in response to rapid pacing, appearing in EPI cells at shorter BCL compared with MID and ENDO cells (BCL=428 +/- 17 vs. 517 +/- 29 and 514 +/- 21, respectively, P < 0.05). Further increases in pacing rate resulted in the appearance of subcellular alternans of Ca(2+) transient amplitude, which also appeared in EPI at shorter BCL than in ENDO and MID cells. Ca(2+) transient alternans was not observed in neonate myocytes. We conclude that 1) there are distinct regional differences in the vulnerability to rate-dependent Ca(2+) alternans in dog LV that may be related to regional differences in SR function and Ca(2+) cycling; 2) the development of subcellular Ca(2+) alternans suggests the presence of intracellular heterogeneities in Ca(2+) cycling; and 3) the failure of neonatal cells to develop Ca(2+) alternans provides further support that SR Ca(2+) cycling is a major component in the development of these phenomena.

Regulation of the Pseudomonas aeruginosa quorum-sensing regulator VqsR.

Bacteria communicate with each other to regulate cell density-dependent gene expression via a quorum-sensing (QS) cascade. In Pseudomonas aeruginosa, two known QS systems, las and rhl, control the expression of many factors that relate to virulence, pathogenicity, and biofilm development. Microarray studies of the las and rhl regulons led to our hypothesis that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. Here, we examined a QS-regulated gene, vqsR, which encodes a probable transcriptional regulator with a putative 20-bp operator sequence (las box) upstream. The transcriptional start site for vqsR was determined. The vqsR promoter was identified by examining a series of vqsR promoter-lacZ fusions. In addition, an Escherichia coli system where either LasR or RhlR protein was expressed from a plasmid indicated that the las system was the dominant regulator for vqsR. Electrophoretic mobility shift assays (EMSA) demonstrate that purified LasR protein binds directly to the vqsR promoter in the presence of 3O-C12-HSL. Point mutational analysis of the vqsR las box suggests that positions 3 and 18 in the las box are important for vqsR transcription, as assayed with a series of vqsRp-lacZ fusions. EMSA also shows that positions 3 and 18 are important for binding between the vqsR promoter and LasR. Our results demonstrate that the las system directly regulates vqsR, and certain nucleotides in the las box are crucial for LasR binding and activation of the vqsR promoter.