Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring.

Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p < 0.001), further indicated a strong relationship between the methods results. From the 361 samples, 185 split samples with ddPCR and Enterolert values within the limits of quantification, were used as a 'training' data set to derive an intrinsic copy number equation (ICE) for scaling ddPCR gene copy number to Enterolert most probable number (MPN). Of the 1993 samples that comprised the complete 'test' data set assayed by ddPCR and Enterolert, 1086 generated results that fell within the limits of quantification for Enterolert and yielded an overall IA of 0.64. Re-analysis using median as a measure of central tendency to account for significant skewing of Enterolert data yielded an IA of 0.72. Beach grouping-specific IA values ranged from 0.63 to 0.93. Pearson's correlation coefficient, r, ranged from 0.13 to 0.94 within beach groupings and generated a combined value of 0.60 for all groupings. Using the ICE, a ddPCR advisory threshold of 1413 DNA copy number/100 mL was empirically determined to be the equivalent to the California Enterolert beach action threshold of 104 MPN/100 mL, based on comparison with all 1993 paired ddPCR and Enterolert results. Using the 1413 DNA copy number/100 mL as a beach action threshold for ddPCR resulted in a 90.4% agreement with Enterolert (6.0% false negative and 3.7% false positive). Together these findings support the conclusion that ddPCR readouts align closely with Enterolert MPN for identifying FIB exceedance levels of Enterococcus spp. in coastal waters of San Diego, CA.

Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells.

Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8+ T cells and type I IFN signaling. Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8+ T cells and led to a synergistic increase in total and tumor antigen-specific CD8+ T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8+ T cells into CD127lowKLRG1high short-lived effector cells, preferentially expanding the CD127highKLRG1low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8+ T cells.

RNA recognition by human TLR8 can lead to autoimmune inflammation.

Studies on the role of the RNA receptor TLR8 in inflammation have been limited by its different function in human versus rodents. We have generated multiple lines of transgenic mice expressing different levels of human TLR8. The high copy number chimeras were unable to pass germline; developed severe inflammation targeting the pancreas, salivary glands, and joints; and the severity of the specific phenotypes closely correlated with the huTLR8 expression levels. Mice with relatively low expression levels survived and bred successfully but had increased susceptibility to collagen-induced arthritis, and the levels of huTLR8 correlated with proinflammatory cytokines in the joints of the animals. At the cellular level, huTLR8 signaling exerted a DC-intrinsic effect leading to up-regulation of co-stimulatory molecules and subsequent T cell activation. A pathogenic role for TLR8 in human diseases was suggested by its increased expression in patients with systemic arthritis and the correlation of TLR8 expression with the elevation of IL-1ß levels and disease status. We found that the consequence of self-recognition via TLR8 results in a constellation of diseases, strikingly distinct from those related to TLR7 signaling, and points to specific inflammatory diseases that may benefit from inhibition of TLR8 in humans.

Chk1 is essential for tumor cell viability following activation of the replication checkpoint.

Chk1 (checkpoint kinase 1) is an evolutionarily conserved serine/threonine kinase involved in DNA damage responses. Originally identified as a kinase regulating the G2/M transition checkpoint, its role has broadened to include the S-phase checkpoint response and essential functions in early embryonic development. In this manuscript we investigated the potential of chemo-sensitization via ablation of Chk1 in cells treated with anti-metabolite cancer drugs, hydroxyurea (HU) and cytosine arabinoside (ara-C). Exposure to these replication interfering drugs in cells carrying Chk1 targeted siRNA provoked markedly increased rates of apoptosis. Although cell death was accompanied by an increase in p53 and activation of Chk2, the increased susceptibility to apoptosis was not dependent on p53 or Chk2. Additionally, we found that cells with reduced Chk1 expression displayed increased gamma-H2A.X expression, a marker for damaged DNA, and phosphorylated 32kDa subunit of replication protein A (RPA). Thus, Chk1 may play an essential role in maintaining DNA integrity during the replication block. Significantly, normal cells such as WS1 did not exhibit increased DNA damage or subsequent increases in apoptosis following replication stress, in the absence of Chk1. Thus, the essential role Chk1 plays in maintaining viability during the replication block in cancer cell lines can be exploited to sensitize cancer cells when abrogation of Chk1 is combined with DNA anti-metabolite chemotherapeutic drugs. Taken together, these data suggest that inhibition of Chk1 in combination with DNA anti-metabolite chemotherapy is a viable therapeutic strategy.

Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation.

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4(+) T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.

In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines.

We show that a combination of the immunosuppressive drugs, vitamin D3 and Dexamethasone, induced human and mouse naive CD4(+) T cells to differentiate in vitro into regulatory T cells. In contrast to the previously described in vitro derived CD4(+) T cells, these cells produced only interleukin (IL)-10, but no IL-5 and interferon (IFN)-gamma, and furthermore retained strong proliferative capacity. The development of these IL-10-producing cells was enhanced by neutralization of the T helper type 1 (Th1)- and Th2-inducing cytokines IL-4, IL-12, and IFN-gamma. These immunosuppressive drugs also induced the development of IL-10-producing T cells in the absence of antigen-presenting cells, with IL-10 acting as a positive autocrine factor for these T cells. Furthermore, nuclear factor (NF)-kappaB and activator protein (AP)-1 activities were inhibited in the IL-10-producing cells described here as well as key transcription factors involved in Th1 and Th2 subset differentiation. The regulatory function of these in vitro generated IL-10-producing T cells was demonstrated by their ability to prevent central nervous system inflammation, when targeted to the site of inflammation, and this function was shown to be IL-10 dependent. Generating homogeneous populations of IL-10-producing T cells in vitro will thus facilitate the use of regulatory T cells in immunotherapy.