Evaluating and Assessing Radon Testing in Multifamily Housing.

CONTEXT: To determine if radon mitigation is needed to protect occupants of multifamily housing, reliable testing procedures are needed. Yet, protocols on how many ground-contact housing units must be tested vary from 10% to 25% to 100%. OBJECTIVES: To estimate the probability of failing to identify a building containing at least one unit with elevated radon level when all ground-contact units are not tested. DESIGN: Retrospective analysis of previously collected data from licensed (ie, certified) radon measurement professionals using hypergeometric and Monte Carlo statistical methods to estimate the confidence that there are no units with radon levels of 4 picoCuries/liter of air (pCi/L) or more based on various testing percentages. SETTING: Testing data were obtained from 29 US states for 7892 ground-contact units in 687 multifamily buildings, primarily 5 to 20 units per building. MAIN OUTCOME MEASURE: Probability of failing to identify elevated radon levels in untested units. RESULTS: About 15% (n = 1163) of the units had radon levels of more than 4 pCi/L (the EPA action level); 59 units had more than 20 pCi/L (maximum of 96 pCi/L). For building sizes of 5 to 20 ground-contact units, the 2018 federal testing protocols that currently require testing of 10% and 25% of ground-contact units in each building failed to identify 47%-69% and 32%-46% of the units, respectively, depending on building size. CONCLUSIONS: Measurement of 90% of the ground-contact units in buildings with 5 to 20 ground-contact units results in up to 4% of the units with elevated radon levels being missed. To achieve 95% confidence that no units in the building have radon levels of 4 pCi/L or more in buildings up to 20 units, 100% sampling is required. For the vast majority of multifamily buildings, all ground-contact units in multifamily buildings should be tested for radon.

Combination Immunotherapy with Passive Antibody and Sulfasalazine Accelerates Fungal Clearance and Promotes the Resolution of Pneumocystis-Associated Immunopathogenesis.

The pulmonary immune response protects healthy individuals against Pneumocystis pneumonia (PcP). However, the immune response also drives immunopathogenesis in patients who develop severe PcP, and it is generally accepted that optimal treatment requires combination strategies that promote fungal killing and also provide effective immunomodulation. The anti-inflammatory drug sulfasalazine programs macrophages for enhanced Pneumocystis phagocytosis and also suppresses PcP-related immunopathogenesis. Anti-Pneumocystis antibody opsonizes Pneumocystis organisms for greater phagocytosis and may also mask antigens that drive immunopathogenesis. Thus, we hypothesized that combining antibody and sulfasalazine would have the dual benefit of enhancing fungal clearance while dampening immunopathogenesis and allow the rescue of severe PcP. To model a clinically relevant treatment scenario in mice, therapeutic interventions were withheld until clear symptoms of pneumonia were evident. When administered individually, both passive antibody and sulfasalazine improved pulmonary function and enhanced Pneumocystis clearance to similar degrees. However, combination treatment with antibody and sulfasalazine produced a more rapid improvement, with recovery of body weight, a dramatic improvement in pulmonary function, reduced lung inflammation, and the rapid clearance of the Pneumocystis organisms. Accelerated fungal clearance in the combination treatment group was associated with a significant increase in macrophage phagocytosis of Pneumocystis Both passive antibody and sulfasalazine resulted in the suppression of Th1 cytokines and a marked increase in lung macrophages displaying an alternatively activated phenotype, which were enhanced by combination treatment. Our data support the concept that passive antibody and sulfasalazine could be an effective and specific adjunctive therapy for PcP, with the potential to accelerate fungal clearance while attenuating PcP-associated immunopathogenesis.

Ataxia-telangiectasia mutated is required for the development of protective immune memory after influenza A virus infection.

Ataxia-telangiectasia (A-T), caused by mutations in the A-T mutated (ATM) gene, is a neurodegenerative disorder affecting ∼1 in 40,000-100,000 children. Recurrent respiratory infections are a common and challenging comorbidity, often leading to the development of bronchiectasis in individuals with A-T. The role of ATM in development of immune memory in response to recurrent respiratory viral infections is not well understood. Here, we infect wild-type (WT) and Atm-null mice with influenza A virus (IAV; HKx31, H3N2) and interrogate the immune memory with secondary infections designed to challenge the B cell memory response with homologous infection (HKx31) and the T cell memory response with heterologous infection (PR8, H1N1). Although Atm-null mice survived primary and secondary infections, they lost more weight than WT mice during secondary infections. This enhanced morbidity to secondary infections was not attributed to failure to effectively clear virus during the primary IAV infection. Instead, Atm-null mice developed persistent peribronchial inflammation, characterized in part by clusters of B220+ B cells. Additionally, levels of select serum antibodies to hemagglutinin-specific IAV were significantly lower in Atm-null than WT mice. These findings reveal that Atm is required to mount a proper memory response to a primary IAV infection, implying that vaccination of children with A-T by itself may not be sufficiently protective against respiratory viral infections.

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.

Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection.

Invasive fungal infections, including Pneumocystis Pneumonia (PcP), remain frequent life-threatening conditions of patients with adaptive immune defects. While innate immunity helps control pathogen growth early during infection, it is typically not sufficient for complete protection against Pneumocystis and other human fungal pathogens. Alveolar macrophages (AM) possess pattern recognition molecules capable of recognizing antigenic and structural determinants of Pneumocystis. However, this pathogen effectively evades innate immunity to infect both immunocompetent and immunosuppressed hosts, albeit with differing outcomes. During our studies of mouse models of PcP, the FVB/N strain was identified as unique because of its ability to mount a protective innate immune response against Pneumocystis infection. In contrast to other immunocompetent strains, which become transiently infected prior to the onset of adaptive immunity, FVB/N mice rapidly eradicated Pneumocystis before an adaptive immune response was triggered. Furthermore, FVB/N mice remained highly resistant to infection even in the absence of functional T cells. The effector mechanism of innate protection required the action of functional alveolar macrophages, and the adoptive transfer of resistant FVB/N AMs, but not susceptible CB.17 AMs, conferred protection to immunodeficient mice. Macrophage IFNγ receptor signaling was not required for innate resistance, and FVB/N macrophages were found to display markers of alternative activation. IFNγ reprogrammed resistant FVB/N macrophages to a permissive M1 biased phenotype through a mechanism that required direct activation of the macrophage IFNγR. These results demonstrate that appropriately programmed macrophages provide protective innate immunity against this opportunistic fungal pathogen, and suggest that modulating macrophage function may represent a feasible therapeutic strategy to enhance antifungal host defense. The identification of resistant and susceptible macrophages provides a novel platform to study not only the mechanisms of macrophage-mediated antifungal defense, but also the mechanisms by which Pneumocystis evades innate immunity.

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.

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.

Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.