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Beethoven's String Quintet, Op. 29, has been described as a ‘wallflower’ work that, without enough suitors, remains on the sidelines of the string chamber music repertoire. But in the nineteenth century it had a prominent champion, Joseph Joachim, whose performances of the quintet must have attracted the attention of his close friend, Johannes Brahms. The opening theme of Brahms's String Sextet, Op. 18, is clearly reminiscent of the beginning of Beethoven's quintet. Evidence from Donald Francis Tovey's recollections of Joachim, Joachim's correspondence with the Brahms biographer Max Kalbeck, and the manuscript of Op. 18 shows that Joachim influenced an important revision that aligns the beginning of Brahms's sextet closely with the opening of Beethoven's Op. 29 also in terms of texture and formal design.The striking tremolo opening and virtuosic scale passages in the finale of Beethoven's quintet prefigure similar elements in the last movement of Brahms's Op. 36 sextet. But the deeper relationship between these movements lies in certain shared formal elements: a common emphasis on sound, texture and sharp contrasts between agitato and pastoral elements as defining features of the overall form – and several distinctive similarities of contrapuntal strategy, form and tonal design between the substantial fugatos that dominate the development sections of both movements.It is often observed that Brahms wrote chamber works in pairs. Scholars have often posited that his two string sextets form such a pair, but the separation of four years in their inceptions and his extensive use of Baroque-style materials composed in the 1850s in the later sextet have made this argument tenuous. It now emerges that an unusual pairing feature of Brahms's string sextets is that both works respond to Beethoven's ‘wallflower’ masterpiece.

The human immune response to inactivated influenza vaccine is dynamic and impacted by age and preexisting immunity. Our goal was to identify postvaccination transcriptomic changes in peripheral blood mononuclear cells from children. Blood samples were obtained before and at 3 or 7 days postvaccination with 2016-2017 quadrivalent inactivated influenza vaccine and RNA sequencing was performed. There were 1,466 differentially expressed genes (DEGs) for the Day 0-Day 3 group and 513 DEGs for the Day 0-Day 7 group. Thirty-three genes were common between the two groups. The majority of the transcriptomic changes at Day 3 represented innate inflammation and apoptosis pathways. Day 7 DEGs were characterized by activation of cellular processes, including the regulation of cytoskeleton, junctions, and metabolism, and increased expression of immunoglobulin genes. DEGs at Day 3 were compared between older and younger children revealing increased inflammatory gene expression in the older group. Vaccine history in the year prior to the study was characterized by robust DEGs at Day 3 with decreased phagosome and dendritic cell maturation in those who had been vaccinated in the previous year. PBMC responses to inactivated influenza vaccination in children differed significantly by the timing of sampling, patient age, and vaccine history. These data provide insight into the expected molecular pathways to be temporally altered by influenza vaccination in children.

Background Endocrine therapy resistance is a hallmark of advanced estrogen receptor (ER)-positive breast cancer. In this study, we aimed to determine acquired genomic changes in endocrine-resistant disease. Methods We performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen deprivation and identified acquired genomic changes versus each tumor’s matched primary. Results Despite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1, and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common—including outlier gains in TP63 (n = 5 cases [42%]), NTRK3 (n = 5 [42%]), NTRK2 (n = 4 [33%]), PAX3 (n = 4 [33%]), FGFR4 (n = 3 [25%]), and TERT (n = 3 [25%]). Recurrent losses involved ESR1 (n = 5 [42%]), RELN (n = 5 [42%]), SFRP4 (n = 4 [33%]), and FOSB (n = 4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers. Conclusions Taken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease; highlights the importance of longitudinal RNA profiling; and identifies a common ESR1-depleted endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.

Background/aims Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins. Methods We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis. Results Single nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium, Man1a2 knockdown arrested ciliary development and motility. Man1a2 -/- mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and Man1a2 -/- liver exhibited reduced Man1a2 expression and dysregulated ciliary genes, known to cause multisystem human laterality defects. Conclusion BA requiring transplantation associates with sequence variants in MAN1A2. man1a2 regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.

SUMMARY Clinical definitions of asthma fail to capture the heterogeneity of immune dysfunction in severe, treatment-refractory disease. Applying mass cytometry and machine learning to bronchoalveolar lavage (BAL) cells, we find that corticosteroid-resistant asthma patients cluster largely into two groups: one enriched in interleukin (IL)-4+ innate immune cells and another dominated by interferon (IFN)-γ+ T cells, including tissue-resident memory cells. In contrast, BAL cells of a healthier population are enriched in IL-10+ macrophages. To better understand cellular mediators of severe asthma, we developed the Immune Cell Linkage through Exploratory Matrices (ICLite) algorithm to perform deconvolution of bulk RNA sequencing of mixed-cell populations. Signatures of mitosis and IL-7 signaling in CD206−FcεRI+CD127+IL-4+ innate cells in one patient group, contrasting with adaptive immune response in T cells in the other, are preserved across technologies. Transcriptional signatures uncovered by ICLite identify T-cell-high and T-cell-poor severe asthma patients in an independent cohort, suggesting broad applicability of our findings., Graphical abstract, In brief Asthma is an immune-mediated heterogeneous disease. Current type-2 biomarkers provide limited understanding of underlying pathobiology or therapeutic guidance. Using a multi-omics approach, Camiolo et al. identify two clusters of severe asthma patients with similar type-2 biomarkers but with strikingly distinct immune profiles and associated biological pathways.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown. Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1–regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease. Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro–computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1–regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort. Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1–regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function. Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1–deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including autoimmunity and IL-17 driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17 mediated cytokine production is unknown. To examine the role of FSTL-1 in IL-17 signaling, we utilized parallel in vitro bone marrow stromal cell models of FSTL-1 attenuation. Microarray identified FSTL-1 regulated genes that could influence IL-17 dependent production of IL-6 and G-CSF, wherein we discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. In FSTL-1 KO BMSCs, ectopic Il17rc expression rescued IL-17 stimulated cytokine, and ectopic Fstl1 expression rescued Il17rc expression as well as IL-17 stimulated cytokines. Mechanisms influencing FSTL-1 dependent Il17rc transcript abundance were examined using Actinomycin D and nascent mRNA labeling approaches and identified that FSTL-1 regulates Il17rc gene transcription. Taken together, this work identifies a mechanism by which FSTL-1 influences IL-17 driven inflammation and reveals a novel function for FSTL-1 as a modulator of gene expression. Thus, enhanced understanding of the interplay between FSTL-1 and IL-17 mediated inflammation may provide insight into potential therapeutic targets of IL-17 mediated diseases.

The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis. These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis., Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivo. Gsc1, encoding a β-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis. GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina. Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of Pneumocystis using a cohousing model. IMPORTANCE The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis. These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis.

Objective To develop a method to perform multiple tests on single nasopharyngeal (NP) swab. Study design We collected a NP swab on children aged 2–12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA sequencing, which we accomplished by cutting the tip of the swab. Results Of the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121(69.5%) tested positive for a virus. Cytokine measurement, as judged by the adequate levels of a housekeeping enzyme (GAPDH), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2,646 operational taxonomic units across all samples sequenced. Samples used for RNA sequencing had a mean RNA Integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis. Conclusion We describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

Rationale: Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4+ T cell–dependent eosinophilic response in the lung capable of providing protection.Objectives: We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection.Methods: Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4+ T cells and eosinophils were examined using antibody depletion and knockout mice, respectively. The presence of anti-Pneumocystis antibodies in human serum samples was detected by ELISA and Western blotting.Measurements and Main Results: Pneumocystis infection generates a strong type II response in the lung that requires CD4+ T cells. Pneumocystis infection was capable of priming a Th2 response similar to th...

RNA sequencing is a method of transcriptome profiling that utilizes next-generation sequencing technology. It offers several distinct advantages over hybridization-based approaches, most notably superior sensitivity and the capacity for de novo transcript discovery. This article describes RNA sequencing methodology, summarizes important technological advances and challenges, and discusses applications for this technique in the field of dermatology.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout ( Il22Ra1 Hep−/− ) and Stat3 knockout ( Stat3 Hep −/− ) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae , which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3 . Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae , which was dependent on lipocalin 2 (LCN2). However, in vivo , rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti- K. pneumoniae bactericidal activity in vitro . These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

Pancreatic ductal adenocarcinoma (PDAC) continues to be an aggressive disease with an immunosuppressive tumor microenvironment, which is recalcitrant to most therapies. Interleukin-17 (IL-17), a potent pro-inflammatory cytokine, has been implicated in initiation of pancreatic premalignant lesions but its role in invasive PDAC has not been widely explored. We hypothesized that IL-17 triggers and sustains immunosuppression during PDAC. We employed pharmacological and genetic strategies to inhibit IL-17/IL-17RA signaling axis and characterized the tumor microenvironment with RNA sequencing, mass & flow cytometry and quantitative multiplex immunofluorescence. Our analysis revealed that IL-17 signaled through tumor cells to recruit neutrophils into the tumor microenvironment and spatially exclude cytotoxic CD8 T cells. Furthermore, IL-17 signaling in tumor cells triggered neutrophils to form extracellular traps (NETs), a primary mechanism mediating PDAC immunosuppression. Genetic or pharmacological inhibition of IL-17 overcame resistance to immune checkpoint blockade (PD-1, CTLA-4), in a CD8 T cell dependent manner. Inhibition of neutrophils or Padi4-dependent NETosis phenocopied IL-17 neutralization. Higher expression of IL-17 and PADI4 in human PDAC corresponded with poorer prognosis, according to TCGA data. Serum from PDAC patients had a higher capacity for NET formation and diminished ability for NET degradation in control neutrophils, in comparison to serum from healthy donors. In conclusion, the cross-talk of IL-17 with pancreatic cancer cells orchestrates neutrophil recruitment and activation of Padi4 dependent NETosis, ultimately resulting in spatial remodeling of the stroma and immunosuppression. IL-17 inhibition overcomes resistance to checkpoint blockade in a CD8 T cell dependent fashion. Clinical studies with IL-17 inhibitors and checkpoint blockade represent a promising novel combinatorial therapy for this lethal disease. Citation Format: Vidhi Chandra, Yu Zhang, Erick Riquelme, Prasanta Dutta, Pompeyo R. Quesada, Amanda Rakoski, Michelle Zoltan, Nivedita Arora, Seyda Baydogan, William Horne, Jared Burks, Hanwen Xu, Perwez Hussain, Huamin Wang, Sonal Gupta, Anirban Maitra, Jennifer M. Bailey, Seyed J. Moghaddam, Sulagna Banerjee, Ismet Sahin, Pratip Bhattacharya, Florencia McAllister. Interleukin 17 induced- neutrophil extracellular traps (NETs) mediate resistance to checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-014.

Background The reasons for differences in vaccine effectiveness between live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are not clear. Methods Blood samples were obtained before vaccination and at days 7 and 21 postvaccination with 2015–2016 quadrivalent IIV or LAIV. Serologic response to the vaccine was measured by hemagglutination inhibition assay. Targeted RNA sequencing and serum cytokine analysis were performed. Paired analyses were used to determine gene expression and were compared between IIV and LAIV recipients. Classification And Regression Trees analysis (CART) identified the strongest associations with vaccine response. Results Forty-six enrollees received IIV, and 25 received LAIV. The mean age was 11.5 (±3.7) years. Seroconversion with IIV was associated with changes in expression of PRKRA and IFI6. Nonseroconversion for both IIV and LAIV was characterized by increased interferon-stimulated gene expression. Seroprotection with both vaccines was associated with altered expression of CXCL2 and CD36. For LAIV, CART showed that changes in expression of CD80, CXCL2, and CASP1 were associated with seroprotection. Serum cytokines showed that IIV seroconversion was associated with decreased CCL3. LAIV seroprotection tracked with decreased tumor necrosis factor–α and interferon-γ. Conclusions Distinct markers of seroconversion and seroprotection against IIV and LAIV were identified using immunophenotyping and CART analysis.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

BACKGROUND & AIMS: Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17 A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to PanIN initiation and progression. We investigated the effects of IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. METHODS: We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KC(iMist);G) and Kras(G12D); Trp53(R172H); Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop pancreatic intraepithelial neoplasias, PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KC(iMist) mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of doublecortin like kinase 1 (DCLK1) and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or small hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using the Cancer Genome Atlas database. RESULTS: PanIN cells from KC(iMist);G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17 neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern, and significantly decreased expression of DCLK1 and POU class 2 homeobox 3 (POU2F3), which regulate tuft cell development. KC(iMist) mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and MAPK signaling, and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells), compared to cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 following incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C (IL17RC) was higher in DCLK1-positive PanIN cells from mice compared to DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 also associated with patient survival time. CONCLUSIONS: In studies of mouse and human pancreatic tumors and precursors, we found immune cell-derived IL17 to regulate development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4+ T cell count in the setting of HIV/AIDS or primary immunodeficiency. Non–HIV-infected individuals taking immunosuppressive drug regimens targeting T cell activation are also susceptible. Given the crucial role of CD4+ T cells in host defense against Pneumocystis, we used RNA sequencing of whole lung early in infection in wild-type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild-type mice, a strong eosinophil signature was observed at day 14 post Pneumocystis challenge, and eosinophils were increased in the bronchoalveolar lavage fluid of wild-type mice. Furthermore, eosinophilopoiesis-deficient Gata1tm6Sho/J mice were more susceptible to Pneumocystis infection when compared with BALB/c controls, and bone marrow–derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1−/− mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. The pIL5-treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden, compared with mice treated with control plasmid. In addition, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57BL/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1tm6Sho/J mice. Taken together, these results demonstrate that an early role of CD4+ T cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development in the treatment of Pneumocystis pneumonia in the immunosuppressed population.

Acute ethanol intoxication suppresses the host immune responses against Streptococcus pneumoniae . As interleukin 17 (IL-17) is a critical cytokine in host defense against extracellular pathogens, including S. pneumoniae , we hypothesized that ethanol impairs mucosal immunity against this pathogen by disrupting IL-17 production or IL-17 receptor (IL-17R) signaling. A chronic ethanol feeding model in simian immunodeficiency virus (SIV)-infected rhesus macaques and acute ethanol intoxication in a murine model were used. Transcriptome analysis of bronchial brushes in the nonhuman primate model showed downregulation of the expression of IL-17-regulated chemokines in ethanol-fed animals, a finding also replicated in the murine model. Surprisingly, recombinant CXCL1 and CXCL5 but not IL-17 or IL-23 plus IL-1β rescued bacterial burden in the ethanol group to control levels. Taken together, the results of this study suggest that ethanol impairs IL-17-mediated chemokine production in the lung. Thus, exogenous luminal restoration of IL-17-related chemokines, CXCL1 and CXCL5, improves host defenses against S. pneumoniae .

Bone metastases (BoM) are a significant cause of morbidity in patients with estrogen receptor–positive (ER-positive) breast cancer; yet, characterizations of human specimens are limited. In this study, exome-capture RNA sequencing (ecRNA-seq) on aged (8–12 years), formalin-fixed, paraffin-embedded (FFPE), and decalcified cancer specimens was evaluated. Gene expression values and ecRNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared with matched flash-frozen or nondecalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matched synchronous or recurrent BoMs. Overtime, BoMs exhibited gene expression shifts to more Her2 and LumB PAM50 subtype profiles, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets, and longitudinal expression alterations of clinically actionable genes, particularly in the CDK/Rb/E2F and FGFR signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines recurrent longitudinal transcriptional remodeling events in estrogen-deprived breast cancers.

Inhibitory receptors are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability and function. However, the role of Inhibitory receptors on regulatory T (Treg) cells remains obscure, as they could be required for suppressive activity and/or limit Treg cell function. We evaluated the role of Lymphocyte Activation Gene-3 (LAG3, CD223) on Treg cells by generating mice in which LAG3 is absent on the cell surface of Treg cells in a murine model of Type 1 Diabetes. Surprisingly, mice that lacked LAG3 expression on Treg cells exhibited reduced autoimmune diabetes, consistent with enhanced Treg cell proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Treg cells was largely comparable, substantial differences between intra-islet Treg cells were evident and involved a subset of genes and pathways that promote Treg cell maintenance and function. Consistent with these observations, Lag3-deficient Treg cells out-competed WT Treg cells in the islets but not in the periphery in co-transfer experiments due to enhanced IL2-Stat5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg cell proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment and may contribute to Treg cell insufficiency in autoimmune disease.

Bone metastases (BoM) are a significant cause of morbidity in patients with Estrogen-receptor (ER)-positive breast cancer, yet characterizations of human specimens are limited. In this study, exome-capture RNA-sequencing (ecRNA-seq) on aged (8-12 years), formalin-fixed paraffin-embedded (FFPE) and decalcified cancer specimens was first evaluated. Gene expression values and RNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared to matched flash-frozen or non-decalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matchedde novoor recurrent BoM. BoMs harbored shifts to more Her2 and LumB PAM50 intrinsic subtypes, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets and altered expression of clinically actionable genes, particularly in the CDK-Rb-E2F and FGFR-signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines expression-based tumor evolution in long-term, estrogen-deprived metastases that may have immediate clinical implications.Grant SupportResearch funding for this project was provided in part by a Susan G. Komen Scholar award to AVL and to SO, the Breast Cancer Research Foundation (AVL and SO), the Fashion Footwear Association of New York, the Magee-Women’s Research Institute and Foundation, and through a Postdoctoral Fellowship awarded to RJW from the Department of Defense (BC123242). NP was supported by a training grant from the NIH/NIGMS (2T32GM008424-21) and an individual fellowship from the NIH/NCI (5F30CA203095).Conflicts of Interest DisclosureNo relevant conflicts of interest disclosed for this study.Author ContributionsStudy concept and design (NP, RJW, SO, AVL); acquisition, analysis, or interpretation of data (all authors); drafting of the manuscript (NP, RJW, SO, AVL); critical revision of the manuscript for important intellectual content (all authors); administrative, technical, or material support (PCL, AB, RB, KRW, WH, JK, MR, ZF, AMB).

Objective To determine whether treatment for urinary tract infections in children could be individualized using biomarkers for acute pyelonephritis. Study design We enrolled 61 children with febrile urinary tract infections, collected blood and urine samples, and performed a renal scan within 2 weeks of diagnosis to identify those with pyelonephritis. Renal scans were interpreted centrally by 2 experts. We measured inflammatory proteins in blood and urine using LUMINEX or an enzyme-linked immunosorbent assay. We evaluated serum RNA expression using RNA sequencing in a subset of children. Finally, for children with Escherichia coli isolated from urine cultures, we performed a polymerase chain reaction for 4 previously identified virulence genes. Results Urinary markers that best differentiated pyelonephritis from cystitis included chemokine (C-X-C motif) ligand (CXCL)1, CXCL9, CXCL12, C-C motif chemokine ligand 2, INF γ, and IL-15. Serum procalcitonin was the best serum marker for pyelonephritis. Genes in the interferon-γ pathway were upregulated in serum of children with pyelonephritis. The presence of E coli virulence genes did not correlate with pyelonephritis. Conclusions Immune response to pyelonephritis and cystitis differs quantitatively and qualitatively; this may be useful in differentiating these 2 conditions.

SUMMARY Interleukin (IL)-17 signaling to the intestinal epithelium regulates the intestinal microbiome. Given the reported links between intestinal dysbiosis, bacterial translocation, and liver disease, we hypothesize that intestinal IL-17R signaling plays a critical role in mitigating hepatic inflammation. To test this, we study intestinal epithelium-specific IL-17RA-deficient mice in an immune-driven hepatitis model. At the naive state, these mice exhibit microbiome dysbiosis and increased translocation of bacterial products (CpG DNA), which drives liver IL-18 production. Upon disease induction, absence of enteric IL-17RA signaling exacerbates hepatitis and hepatocyte cell death. IL-18 is necessary for disease exacerbation and is associated with increased activated hepatic lymphocytes based on Ifng and Fasl expression. Thus, intestinal IL-17R regulates translocation of TLR9 ligands and constrains susceptibility to hepatitis. These data connect enteric Th17 signaling and the microbiome in hepatitis, with broader implications on the effects of impaired intestinal immunity and subsequent release of microbial products observed in other extra-intestinal pathologies., In Brief Castillo-dela Cruz et al. describe a unique protective role of intestinal IL-17RA in hepatitis. Disruption of intestinal IL-17RA signaling results in microbiome dysbiosis and translocation of bacterial products, specifically unmethylated CpG DNA, to the liver. This promotes IL-18 production and subsequent lymphocyte activation and cell death to exacerbate liver inflammation., Graphic Abstract

Regulatory T cells (T

Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to Pseudomonas aeruginosa infection. Recent data suggest that IL-17 contributes to pathological inflammation in the setting of abnormal mucosal immunity, and type 17 immunity–driven inflammatory responses may represent a target to block aberrant inflammation in CF. Indeed, transcriptomic analysis of the airway epithelium from CF patients undergoing clinical bronchoscopy revealed upregulation of IL-17 downstream signature genes, implicating a substantial contribution of IL-17–mediated immunity in CF lungs. Bromodomain and extraterminal domain (BET) chromatin modulators can regulate T cell responses, specifically Th17-mediated inflammation, by mechanisms that include bromodomain-dependent inhibition of acetylated histones at the IL17 locus. Here, we show that, in vitro, BET inhibition potently suppressed Th17 cell responses in explanted CF tissue and inhibited IL-17–driven chemokine production in human bronchial epithelial cells. In an acute P. aeruginosa lung infection murine model, BET inhibition decreased inflammation, without exacerbating infection, suggesting that BET inhibition may be a potential therapeutic target in patients with CF.

There has been a recent trend to increase capability and drive down the Size, Weight and Power (SWAP) of satellites. NASA scientists and engineers across many of NASA's Mission Directorates and Centers are developing exciting CubeSat concepts and welcome potential partnerships for CubeSat endeavors. From a "Telemetry, Tracking and Command (TT&C) Systems and Flight Operations for Small Satellites" point of view, small satellites including CubeSats are a challenge to coordinate because of existing small spacecraft constraints, such as limited SWAP and attitude control, and the potential for high numbers of operational spacecraft. The NASA Space Communications and Navigation (SCaN) Program's Near Earth Network (NEN) and Space Network (SN) are customer driven organizations that provide comprehensive communications services for space assets including data transport between a mission's orbiting satellite and its Mission Operations Center (MOC). This paper presents how well the SCaN networks, SN and NEN, are currently positioned to support the emerging small small satellite and CubeSat market as well as planned enhancements for future support.

The development of a tuberculosis (TB) vaccine that induces sterilizing immunity to Mycobacterium tuberculosis infection has been elusive. Absence of sterilizing immunity induced by TB vaccines may be due to delayed activation of mucosal dendritic cells (DCs), and subsequent delay in antigen presentation and activation of vaccine-induced CD4[superscript +] T-cell responses. Here we show that pulmonary delivery of activated M. tuberculosis antigen-primed DCs into vaccinated mice, at the time of M. tuberculosis exposure, can overcome the delay in accumulation of vaccine-induced CD4[superscript +] T-cell responses. In addition, activating endogenous host CD103[superscript +] DCs and the CD40–CD40L pathway can similarly induce rapid accumulation of vaccine-induced lung CD4[superscript +] T-cell responses and limit early M. tuberculosis growth. Thus, our study provides proof of concept that targeting mucosal DCs can accelerate vaccine-induced T-cell responses on M. tuberculosis infection, and provide insights to overcome bottlenecks in TB vaccine efficacy., National Institutes of Health (U.S.) (grant HL105427), National Institutes of Health (U.S.) (grant AI127172), United States. Army Research Office. Institute for Soldier Nanotechnologies (contract W911NF-13-D-0001), Howard Hughes Medical Institute (Investigator)

Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection.Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4Pneumocystis infection generates a strong type II response in the lung that requires CD4The present study demonstrates for the first time, to our knowledge, that Pneumocystis is an airway allergen capable of inducing asthma-like lung pathology.

Interleukin-17 (IL-17) and IL-17 receptor (IL-17R) signaling are essential for regulating mucosal host defense against many invading pathogens. Commensal bacteria, especially segmented filamentous bacteria (SFB), are a crucial factor that drives T helper 17 (Th17) cell development in the gastrointestinal tract. In this study, we demonstrate that Th17 cells controlled SFB burden. Disruption of IL-17R signaling in the enteric epithelium resulted in SFB dysbiosis due to reduced expression of α-defensins, Pigr, and Nox1. When subjected to experimental autoimmune encephalomyelitis, IL-17R-signaling-deficient mice demonstrated earlier disease onset and worsened severity that was associated with increased intestinal Csf2 expression and elevated systemic GM-CSF cytokine concentrations. Conditional deletion of IL-17R in the enteric epithelium demonstrated that there was a reciprocal relationship between the gut microbiota and enteric IL-17R signaling that controlled dysbiosis, constrained Th17 cell development, and regulated the susceptibility to autoimmune inflammation.

Despite recent advances in understanding macrophage activation, little is known regarding how human alveolar macrophages in health calibrate its transcriptional response to canonical TLR4 activation. In this study, we examined the full spectrum of LPS activation and determined whether the transcriptomic profile of human alveolar macrophages is distinguished by a TIR-domain-containing adapter-inducing interferon-β (TRIF)-dominant type I interferon signature. Bronchoalveolar lavage macrophages were obtained from healthy volunteers, stimulated in the presence or absence of ultrapure LPS in vitro, and whole transcriptomic profiling was performed by RNA sequencing (RNA-Seq). LPS induced a robust type I interferon transcriptional response and Ingenuity Pathway Analysis predicted interferon regulatory factor (IRF)7 as the top upstream regulator of 89 known gene targets. Ubiquitin-specific peptidase (USP)-18, a negative regulator of interferon α/β responses, was among the top up-regulated genes in addition to IL10 and USP41, a novel gene with no known biological function but with high sequence homology to USP18. We determined whether IRF-7 and USP-18 can influence downstream macrophage effector cytokine production such as IL-10. We show that IRF-7 siRNA knockdown enhanced LPS-induced IL-10 production in human monocyte-derived macrophages, and USP-18 overexpression attenuated LPS-induced production of IL-10 in RAW264.7 cells. Quantitative PCR confirmed upregulation of USP18, USP41, IL10, and IRF7. An independent cohort confirmed LPS induction of USP41 and IL10 genes. These results suggest that IRF-7 and predicted downstream target USP18, both elements of a type I interferon gene signature identified by RNA-Seq, may serve to fine-tune early cytokine response by calibrating IL-10 production in human alveolar macrophages.

Two critiques of Brahms's Variations on an Original Theme, Op. 21, No. 1, written by Joseph Joachim in letters to the composer, show that the form of this work passed through at least three distinct versions. A hitherto overlooked remark by Clara Schumann suggests that the variations were essentially finished by the end of 1858. An examination of the music reveals the influence of Joachim's Variations on an Original Theme, Op. 10, and the variation movement of Beethoven's Piano Sonata, Op. 111, the latter serving as an antecedent especially for Brahms's eleventh variation and coda.

Background In recent influenza seasons, the live attenuated influenza vaccine (LAIV) has not demonstrated the same level of vaccine effectiveness as that observed among children who received the inactivated influenza vaccine (IIV). To better understand this difference, this study compared the mRNA sequencing transcription profile (RNA seq) in children who received either IIV or LAIV. Methods Children 3–17 years of age receiving quadrivalent influenza vaccine were enrolled. Blood samples were collected on Day 0 prior to vaccination and again on Day 7 (range 6–10 days) following vaccination. Total RNA was isolated from PAXgene tubes and sequenced for a custom panel of 89 transcripts using the TruSeq Targeted RNA Expression method. Fold differences in normalized RNA seq counts from Day 0 to Day 7 were calculated, log2 transformed and compared between the two vaccine groups. Results Of 73 children, 47 received IIV and 26 received LAIV. Following IIV vaccination, 12 genes demonstrated significant differential expression at Day 7. In contrast, following LAIV vaccination, seven genes demonstrated significant differential expression at Day 7, five of which were not differentially expressed by IIV. Two genes demonstrated similar patterns of regulation in both IIV and LAIV recipients. Conclusion Differential regulation of genes was observed between 2015 and 2016 LAIV and IIV recipients. These results help to elucidate the immune response to influenza vaccines and might help explain the difference in vaccine effectiveness observed in recent years between LAIV and IIV. Disclosures R. Zimmerman, sanofi pasteur: Grant Investigator, Research grant Merck & Co, Inc.: Grant Investigator, Research grant Pfizer, Inc.: Grant Investigator, Research grant; C. J. Lin, Sanofi: Grant Investigator, Research grant Merck & Co, Inc.: Grant Investigator, Research grant Pfizer, Inc..: Grant Investigator, Research grant; M. P. Nowalk, Merck & Co, Inc.: Grant Investigator, Research grant Pfizer, Inc.: Grant Investigator, Research grant

Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1–/–) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1–/– Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

The incidence of life-threatening disseminated Candida albicans infections is increasing in hospitalized patients, with fatalities as high as 60%. Death from disseminated candidiasis in a significant percentage of cases is due to fungal invasion of the kidney, leading to renal failure. Treatment of candidiasis is hampered by drug toxicity, the emergence of antifungal drug resistance and lack of vaccines against fungal pathogens. IL-17 is a key mediator of defense against candidiasis. The underlying mechanisms of IL-17-mediated renal immunity have so far been assumed to occur solely through the regulation of antimicrobial mechanisms, particularly activation of neutrophils. Here, we identify an unexpected role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in C. albicans-infected kidney, and we show that the KKS provides significant renal protection in candidiasis. Microarray data indicated that Klk1 was upregulated in infected kidney in an IL-17-dependent manner. Overexpression of Klk1 or treatment with bradykinin rescued IL-17RA-/- mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following C. albicans infection. Furthermore, combining a minimally effective dose of fluconazole with bradykinin markedly improved survival compared to either drug alone. These results indicate that IL-17 not only limits fungal growth in the kidney, but also prevents renal tissue damage and preserves kidney function during disseminated candidiasis through the KKS. Since drugs targeting the KKS are approved clinically, these findings offer potential avenues for the treatment of this fatal nosocomial infection., Author Summary Candida albicans is the causative agent of oropharyngeal candidiasis (OPC, thrush), dermal and vaginal candidiasis. However, the most severe C. albicans-induced disease is disseminated candidiasis, a frequent nosocomial infection associated with a high mortality rate. During disseminated candidiasis, C. albicans form invasive hyphae that damage target organs, particularly kidney and liver. Previous studies have identified an essential role of interleukin-17 (IL-17) in controlling systemic infection through regulation of neutrophils. We show here for the first time that IL-17 also regulates the renal protective Kallikrein-kinin system (KKS). Our discovery of a connection between IL-17 and the KKS suggests a new, previously unanticipated avenue for the treatment of renal damage in disseminated candidiasis. These findings have potential translational significance, as agonists of the KKS are in routine clinical use. Therefore, these results not only identify downstream mediators that could serve as novel drug targets, but could possibly be used to guide decisions on whether targeting these mediators could be a useful therapeutic option in conjunction with current antifungal therapies.

The gastrointestinal tract harbors a complex immune system to contend with both normal flora and invasive pathogens. Recently, interleukin (IL)-22 signaling has proven to play a critical role in maintaining the gut barrier, promoting epithelial healing and controlling inflammation from attaching and effacing pathogens. Specifically, IL-22 produced by Th17 cells and group 3 innate lymphoid cells (ILCs) is critical for host resistance to the attaching and effacing gut pathogen Citrobacter rodentium. However, the importance of intestinal epithelial IL-22Ra1 signaling in regulating secretory cell development and protecting against infectious colitis remains unknown. We hypothesize that intestinal IL-22 signaling plays an important role in mucosal host defense against infectious colitis by regulating the intestinal cell types that maintain the gut barrier. To test this hypothesis, we generated mice that selectively lack the receptor for IL-22, IL-22RA1, and discovered these mice have a striking phenotype of intestinal cell differentiation. We demonstrate that abrogation of intestinal IL-22 signaling led to a decreased number of goblet and paneth cells and decreased expression of the antimicrobial peptide Reg3g. Further, the IL-22RA1 intestinal specific knock-out mice were more susceptible to C. rodentium colitis than WT mice as demonstrated by increased weight loss that correlated with increased spleen bacterial burden, decreased epithelial cell proliferation and disrupted mucosal architecture. These findings offer insights into the protective role that IL-22 plays on mediating intestinal mucosal host defense and how critical IL-22 signaling is to intestinal cell differentiation and attenuating infectious colitis.

The title compound, a 1,3-dipolar cycloaddition product, crystallizes in the monoclinic space groupP21/c, witha=8.199(3),b=16.908(1),c=10.248(2)A,β=93.58(2)° andZ=4. The structure was solved by direct methods and refined by full matrix least squares methods toR=0.038 for 1687 observed reflections. The stereochemistry of this compound was found to have the “ee” conformation in the solid state as well as in solution. The piperidine ring in the molecule is in the chair form and the isoxazolidine ring adopts an envelope conformation.

The title compound, [C16H36N]+[C48H30Au3Ag2]−, crystallizes in the triclinic space groupP1−1, witha=18.199(12),b=19.210(8),c=19.270(9)A,α=71.34(3)°β=76.43(4)° γ=72.95(5)° andZ=4, with two independent molecules in the asymmetric unit. The structure was solved by direct methods and refined by full-matrix least squares methods toR=0.072 for 3739 observed reflections. The metal atoms have a trigonal-bipyramidal arrangement with the three Au atoms forming an equilateral triangle and two Ag atoms occupying apical positions. Each Au atom is σ-bonded to phenylethynyl ligands in a linear coordination. Each Ag atom is asymmetrically pi-bonded to three alkyne groups. The Au-Au distances lie in the range of 3.934–4.010 A, indicating the lack of Au-Au bond.