Search

Immune checkpoint blockade has shown impressive efficacy in patients with inflamed tumors, although minimal activity has been observed in tumors lacking T cells. Myeloid cells are one of the most abundant cell types in both inflamed and non-inflamed tumors, and may contribute to immune checkpoint blockade resistance. The plasticity of macrophages enables them to directly and indirectly modulate T cell responses, and directly kill tumor cells via phagocytosis. This suggests that targeting myeloid cells could be an effective therapeutic approach. Class I PI3Ks are a family of dual specificity lipid and protein kinases. Unlike other class I PI3Ks, PI3Kγ is predominantly expressed in myeloid cells. PI3Kγ has been shown to be a key mediator that drives the immunosuppressive macrophage program by stimulating AKT/mTOR signaling and promote C/EBPβ expression while inhibiting NF-кB activity (Keneda MM. Nature. 2016;17:437-442). Here, we present the discovery and characterization of INCB098377, a potent and selective PI3Kγ inhibitor. Specific inhibition of PI3Kγ with INCB098377 may induce anti-tumor activity by reshaping the tumor immune microenvironment. In cell-based assays, INCB098377 has an IC50 of 1.4 nM and is greater than 100-fold selective over other PI3K isoforms. It also shows a favorable PK profile in several animal species. Treatment of M2 polarized macrophages with INCB098377 resulted in changes towards a more pro-inflammatory phenotype. CD163 and CD206 were decreased, whereas HLA-DR and co-stimulatory CD80/86 molecules were increased. MHC-I expression was unchanged, suggesting a role for these macrophages in MHC-II-mediated antigen presentation. Furthermore, INCB098377 treatment reduced macrophage-mediated immunosuppression and restored T cell proliferation in M2 polarized macrophages co-cultured with allogeneic human T cells. In vivo, significant tumor growth inhibition was observed with once-daily dosing of 10 mg/kg INCB098377 in both syngeneic and humanized mouse tumor models without toxicity. Moreover, efficacy was observed in inflamed and non-inflamed tumor models. Consistent with the proposed mechanism of action, INCB098377 inhibited phospho-AKT levels in vivo and in human PBMCs. Treatment with INCB098377 induced pro-inflammatory responses without macrophage depletion suggests that robust tumor microenvironment changes are responsible for observed anti-tumor efficacy. In addition, INCB098377 inhibited neutrophil migration in the Carrageenan-induced paw inflammation model. INCB098377, a potent and selective inhibitor of PI3Kγ, shows effective anti-tumor activity in a variety of mouse and humanized cancer models through the inhibition of immunosuppressive cells trafficking into the tumor, modulation of myeloid cell function, and enhancement of T cell proliferation. Acknowledgments: Diana Alvarez Arias and Stephen Douglass contributed equally to this study. Citation Format: Diana A. Arias, Stephen Douglass, Lisa Truong, Qian Wang, Kathy H. Wang, Gengjie Yang, Michael Hansbury, Sybil O’Connor, Kevin Bowman, Robert Collins, Matthew Stubbs, Leslie Hall, Christina Stevens, Christopher Maddage, Brent Douty, Maryanne Covington, Lynn Leffet, Eddy Yue, Andrew Combs, Sunkyu Kim, Niu Shin, Holly Koblish, Rodrigo Hess. Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5162.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3Kδ inhibitor that differs in structure from first-generation PI3Kδ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3Kδ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3Kδ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3Kδ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds.

Unfortunately, within the extant scholarship that has explored queer and trans* historically Black college and university (HBCU) students, the discourse(s) that deliberately center how they can be retained, persist, and ultimately graduate have largely been absent from the literature. Thus, this conceptual exploration offers strategies that HBCUs can and should utilize to ensure that their queer and trans* students persist and graduate. A practice-oriented model is also presented to serve as a guide for HBCU student affairs practioners, presidents, and faculty members to implement so that they may inculcate environments of “success” for their queer and trans* students. Ultimately, the “Queer and Trans* HBCU Student Engagement and Retention Practice Model” illustrates how HBCUs can engage their queer and trans* students during admissions or recruitment, matriculation, and even as alumni.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

Background MCLA-145 is a CD137 x PD-L1 bispecific antibody that releases PD-L1 mediated T-cell inhibition and activates and expands T cells through agonism of CD137. Immune checkpoint inhibitors (ICI) against PD-(L)1 have demonstrated anti-tumor efficacy in a fraction of patients across a broad range of cancers. CD137 (4-1BB, tumor necrosis factor receptor superfamily 9) is an inducible costimulatory receptor transiently expressed on T cells after TCR engagement. CD137 signaling is triggered by receptor clustering and leads to enhanced cytokine production; T cell proliferation, survival, and effector function; and immunological memory formation. Targeting of PD-L1 and CD137 with MCLA-145 may achieve synergistic activity by simultaneously blocking the inhibitory checkpoint PD-L1 and activating tumor specific T cells through co-stimulation. Methods We performed combinatorial functional screening of bispecific antibodies generated from high affinity inhibitory Fabs binding PD-L1 combined with a large and diverse panel of agonistic CD137 Fabs. Results MCLA-145 was selected based on its in vitro potency in multiple primary human immune cell assays. Further, it displays an ability to reverse T cell suppression mediated by M2 macrophages or Tregs. MCLA-145 binds to a unique epitope in the cysteine rich domain 2 of CD137 that overlaps with the CD137L binding region, and all potent bAbs in the screen were able to bind to this region. MCLA-145 drives activation of CD137 and the degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells. Using proximity ligation assays and confocal microscopy we demonstrated that MCLA-145 clusters CD137 on the surface of T cells resulting in internalization. The binding location of MCLA-145 on CD137 may be optimal for the formation of ‘immunological synapses’ with PD-L1 expressing antigen presenting cells or tumors resulting in the potent activation of tumor specific cytotoxic T cells. Conclusions These experiments demonstrate the dual anti-cancer activity of MCLA-145 in preclinical models: release of T-cell checkpoint inhibition through PD-L1; and activation and expansion of T cells through CD137, therefore overcoming T-cell exhaustion and increasing T-cell presence/activity (infiltration) in tumors. MCLA-145 is currently undergoing clinical development in an ongoing trial (NCT03922204). Ethics Approval Animal experiments were performed according to guidelines for animal care of the local Animal Experiments Committee; Use of human blood cells from healthy volunteers was approved by the blood bank’s Ethical Advisory Council.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)

Bladder cancer is among the most prevalent cancers worldwide, with urothelial carcinoma accounting for approximately 90% of cases. Until recently, poor overall survival after chemotherapy combined with immune checkpoint blockade therapies for patients with advanced urothelial carcinoma highlighted the need for new therapies. Although data supports the use of targeted therapies, such as FGFR inhibitors, recent advances in the treatment of bladder cancer are changing the landscape. Enfortumab vedotin (EV) is an antibody drug conjugate (ADC) consisting of an anti-Nectin-4 antibody conjugated to monomethyl auristatin E, which specifically delivers the auristatin payload to Nectin-4 high-expressing urothelial carcinomas. While activity and durability of response from both monotherapy trials as well as combination with pembrolizumab are favorable, additional treatment options might be required for either patients not responding to treatment or those who might develop resistance. Importantly, FGFR activation through rearrangement or mutation is frequently found in the luminal papillary subtype which also expresses high levels of Nectin-4. Thus, we sought to test the potential of combining pemigatinib and EV in models of human bladder cancer which express both activated FGFR3 and Nectin-4. Our initial analysis show that FGFR3 mutant tumors express high levels of Nectin-4. We further demonstrated that two bladder cancer cell lines RT112/84 (FGFR3-TACC3 fusion) and UM-UC-14 (FGFR3S249C) are sensitive to both pemigatinib and EV in vitro and in vivo. Notably, synergistic anti-tumor effects were observed when pemigatinib was combined with EV in vivo. In addition, combination of pemigatinib with EV significantly improved overall survival when compared to single treatments in these models. Altogether, our data strongly suggest a potential for combination of these therapies in the clinic. Citation Format: Rodrigo A. Hess, Lisa Truong, Antony Chadderton, Michelle Frascella, Leslie Hall, Holly Koblish. Synergistic effect of combination of pemigatinib with enfortumab vedotin (EV) in human bladder cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P245.

BackgroundBlocking the PD-L1 immune checkpoint axis with therapeutic antibodies against either the ligand or PD-1 has proven to be an effective treatment modality for multiple cancer histologies. Small molecules targeting the PD-L1/PD-1 axis represent an alternate modality of blocking this pathway. INCB090244 is a small molecule that blocks the PD-L1/PD-1 interaction and restores T cell function similar to the clinical stage PD-L1 inhibitor INCB086550.MethodsMDA-MB-231 or CHO cells overexpressing PD-L1 were used to investigate effects of INCB090244 on PD-L1 dimerization, and intracellular trafficking. In vivo, CD34+ humanized mice harboring MDA-MB-231 tumors or C57Bl/6 mice bearing GL261 subcutaneous or orthotopic tumors were used to investigate the efficacy, biodistribution, and pharmacodynamic effects of INCB090244. Human specific gene expression changes in tumors from MDA-MB-231 bearing humanized mice were analyzed by RNA sequencing.ResultsIn vitro, INCB090244 potently disrupted the PD-L1:PD-1 interaction, induced PD-L1 dimerization, and inhibited PD-1-mediated negative signaling, resulting in enhanced IFN gamma and IL-2 production in primary human immune cells. Following dimerization, INCB090244 induced internalization of PD-L1 resulting in co-localization with the Golgi apparatus and partial localization in the nucleus. After cell treatment and washing, full restoration of PD-L1 at the cell surface was observed after 5 days of culture in vitro. In vivo, INCB090244 reduced tumor growth in CD34+ humanized mice bearing MDA-MB-231 tumors, to similar levels as atezolizumab. Antitumor activity was completely abrogated in immunodeficient mice, confirming the pharmacologic dependency on a competent immune system. RNA sequencing analysis on tumors from these mice demonstrated similar T cell activation gene signatures as clinical checkpoint blockade antibodies. Biodistribution studies in mice bearing both subcutaneous and orthotopically implanted GL261 glioma tumors demonstrated higher accumulation of INCB090244 in tumor tissue compared to PD-L1 antibodies.ConclusionsINCB090244 effectively disrupted the PD-L1/PD-1 interaction, induced dimerization and internalization of PD-L1, restored immunity in in vitro and in vivo tumor models, and is a suitable surrogate for the clinical candidate INCB086550. RNA sequencing demonstrated T cell activation signatures similar to those observed in patients receiving checkpoint blockade antibodies. Biodistribution studies demonstrated higher subcutaneous and brain tumor penetration by INCB090244 compared to PD-L1 antibodies, suggesting a potential advantage of small molecule PD-L1 inhibitors in accessing intratumoral regions. These data further support the clinical evaluation of small molecule PD-L1 inhibitors as an alternative approach to immune therapy.

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin's lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1β (MIP-1beta) and tumor necrosis factor-β (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin's lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

[Image: see text] A medicinal chemistry effort focused on identifying a structurally diverse candidate for phosphoinositide 3-kinase delta (PI3Kδ) led to the discovery of clinical candidate INCB050465 (20, parsaclisib). The unique structure of 20 contains a pyrazolopyrimidine hinge-binder in place of a purine motif that is present in other PI3Kδ inhibitors, such as idelalisib (1), duvelisib (2), and INCB040093 (3, dezapelisib). Parsaclisib (20) is a potent and highly selective inhibitor of PI3Kδ with drug-like ADME properties that exhibited an excellent in vivo profile as demonstrated through pharmacokinetic studies in rats, dogs, and monkeys and through pharmacodynamic and efficacy studies in a mouse Pfeiffer xenograft model.

An aerodynamic database has been generated for use by the Orion Multi-Purpose Crew Vehicle (MPCV) Program to analyze Service Module (SM) panel jettison from the NASA SLS vehicle. The database is a combination of CFD data for the panel aerodynamic coefficients, and MATLAB code written to query the CFD data. The Cart3D inviscid CFD flow solver was used to generate the panel aerodynamic coefficients for static panel orientations and free stream conditions that can occur during the jettison event. The MATLAB code performs the multivariate interpolation to obtain aerodynamic coefficients. The MATLAB code uses input for SM panel parameters and returns the SM panel aerodynamic force and moment coefficients for use with a Six-Degree-of-Freedom (6-DOF) motion solver to model the jettison event. This paper examines the accuracy of the sequential-static database approach by modeling the panel jettison event with a fully-coupled, time-dependent, viscous, moving-body CFD simulation. The fully-coupled simulation is obtained using the Loci/Chem unstructured Navier-Stokes CFD solver. The results show that the fully-coupled approach agrees well with the loosely-coupled database/6-DOF approach, indicating that unsteady effects are minimal for the panel jettison event. These results suggest that the database/6-DOF approach is sufficient. In addition, this paper presents the development of an uncertainty model for use in Monte Carlo analysis of the panel jettison event. Here viscous CFD simulations are obtained with Loci/Chem and compared to the inviscid CFD forces and moments. An uncertainty model based on model-form error and numerical error is presented.

Phosphatidylinositol 3-kinase delta (PI3K

INTRODUCTION: Benefits of early Highly Active AntiRetroviral Therapy (HAART) to reduce infant mortality and morbidity have been demonstrated in resource-limited and rich settings. However, immunovirological data collected in Sub-Saharan Africa are scarce. This study describes the long-term outcome of South African children who started HAART before one year of age (Early Starters Cohort or ESC) and compare their immunovirological outcomes to children who started their therapy after two years of age (Late Starters Cohort or LSC). Immunovirological results will be compared in order to evaluate the long-term non-inferiority of early treatment initiation. METHODS: Fifty-five children were included in the ESC (mean follow-up period 7.9 years) and 96 children were included in the LSC (mean follow-up period 6.3 years). Children from the ESC and the LSC were subdivided into three subgroups according to CD4+% at HAART initiation (100 cp/ml) was comparable in both cohorts but persistent undetectable viral load (

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. Here we have developed an Fc-silenced bispecific IgG1 antibody to CD137 and PD-L1 with monovalent binding specificity to each target. MCLA-145 drives transactivation of CD137 in the vicinity of cells expressing PD-L1, such as in the immunosuppressive tumor microenvironment. The degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells, as demonstrated in transactivation assays whereby reporter T cells were co-cultured with cells expressing different levels of PD-L1. PD-L1 expression as low as 6000 receptors per cell was sufficient to activate CD137 in neighboring T cells. In contrast, MCLA-145 blocked PD-1 signaling without requirement for CD137 binding in a PD-1/PD-L1 reporter assay. CD137 signaling was induced by MCLA-145 in multiple primary human immune cell assays including the mixed lymphocyte reaction, human PBMC, and whole blood SEB stimulation assays. MCLA-145 reversed T cell suppression mediated by M2 macrophages or Tregs, in vitro. In addition, MCLA-145 enhanced Ag-specific expansion and differentiation of human naïve CD8+ T cells in vitro. In vivo, MCLA-145 treatment resulted in significant tumor immune activation and antitumor responses in two separate humanized mouse tumor models. In one model, human T cells expressing NY-ESO specific TCR were adoptively transferred to mice bearing A549 tumors which expressed NY-ESO antigen and human PD-L1. MCLA-145 treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) as compared to T cell only treated mice. In the tumors of MCLA-145 treated mice, the percentage of NY-ESO specific CD8+ T cells were significantly increased compared to controls. In a second model, mice engrafted with human CD34+ cells were implanted with the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and 5 mg/kg induced significant tumor growth inhibition (55 and 57% respectively) as compared to vehicle control or Fc-silenced huIgG1 controls. Additionally, two out of nine animals in the 5 mg/kg MCLA-145-treated group had complete tumor regression. MCLA-145 increased the number of infiltrating CD8+ T cells, as well as the percentage of central memory CD8+ T cells. The cured animals were then re-challenged with MDA-MB-231 tumor cells, and tumors of previously cured mice were rejected as compared to no growth inhibition in treatment-naïve CD34+ NSG mice. In conclusion, these data support the clinical evaluation of MCLA-145 as a novel, PD-L1 dependent CD137 agonist immune therapy. Citation Format: Patrick Mayes, Paul Tacken, Steve Wang, Pieter-Fokko van Loo, Thomas Condamine, Hans van der Maaden, Eric Rovers, Steef Engels, Floris Fransen, Ashwini Kulkarni, Yao-bin Liu, Arpita Mondal, Leslie Hall, Soyeon Kim, Marina Martinez, Shaun O'Brien, Edmund Moon, Steven Albelda, Peggy Scherle, Gregory Hollis, Reid Huber, Mark Throsby, Cecile A. Geuijen. A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 539.

Emerging fungal infections are an important component of infectious disease diagnosis and public health assessment. These infections cause significant morbidity and mortality and can be difficult to diagnose. Recent episodes of fungal infections are presented, including the ongoing Cryptococcus gattii outbreak in British Columbia, Canada, and the northwestern United States; the Apophysomyces trapeziformis infections associated with the Joplin, Missouri, tornado in 2011; the emerging unusual Aspergillus infections associated with antifungal prophylaxis; and the ongoing fungal meningitis outbreak associated with contaminated steroids. Lessons learned from these episodes are discussed in order to provide a platform for illustrating dilemmas in mycology. Useful tools to help the microbiology laboratory stay informed about emerging fungal infections are also discussed.

The PLEX-ID system is a novel technology that couples PCR amplification and electrospray ionization-mass spectrometry to identify pathogens directly in clinical specimens. The analytical performance of the PLEX-ID Broad Fungal assay was compared with that of traditional culture identification by using 91 characterized fungal culture isolates (64 manufacturer-claimed and 27 nonclaimed organisms) and directly by using 395 respiratory specimens. Discordant results were resolved by D2 large-subunit ribosomal DNA fungal sequencing. Environmental studies were performed to monitor for potential contamination. The PLEX-ID Broad Fungal assay correctly identified 95.6% (87/91) and 81.3% (74/91) of the culture isolates to the genus and species levels, respectively. Of the manufacturer-claimed organisms, 100% (64/64) and 92.2% (59/64) were correctly identified to the genus and species levels, respectively. Direct analysis of respiratory specimens resulted in 67.6% (267/395) and 66.6% (263/395) agreement with culture results to the genus and species levels, respectively, with 16.2% (64/395) of the results discordant with culture and 16.2% (64/395) not detected by the system. The majority (>95%) of the isolates not detected directly by the PLEX-ID system ultimately grew in low quantities in culture (≤20 colonies). In 20.3% (35/172) of the respiratory specimens where no growth was observed in culture, the PLEX-ID system identified a fungus, suggesting a potential increase in sensitivity over culture in some instances. The PLEX-ID system provides a rapid method for the detection of a broad array of fungi directly in respiratory specimens and has the potential of impacting turnaround times and patient care by reducing the need to wait for the growth of an organism in culture.

Prevalence of Human-Immunodeficiency-Virus/Hepatitis-B-virus (HIV/HBV) coinfection and HBV vaccination response in children are unknown in Kwazulu-Natal. This study included 183 HIV-infected and 108 HIV-uninfected children aged between 5 and 15 years screened for HBV infection and vaccination. HBV infection occurred in 2.1% and 0% of HIV-infected and uninfected children respectively. Serological response to immunization was shown in 15.8% and 61.1% of HIV-infected and uninfected children, respectively (P 0.001). Even if prevalence of HBV infection was low in these cohorts, HIV-infected children will stay at risk of infection if the vaccine schedule is not adapted. J. Med. Virol. 89:182-185, 2017. © 2016 Wiley Periodicals, Inc.

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

Perhaps no other technique has revolutionized the identification and characterization of a single clinically relevant genus as much as the use of sequencing for mycobacterial diagnostics. This chapter provides a comprehensive account of the current use of DNA sequencing for the identification and characterization of mycobacteria in the clinical microbiology laboratory. The problems associated with phenotypic identification of mycobacteria were highlighted in 1996, when Springer and colleagues compared 34 isolates identified by both biochemical testing and 16S rRNA sequencing. There are no FDA-approved platforms at this time designed specifically for the sequence-based identification of microorganisms. However, there are a number of commercially available sequencing platforms, and laboratories are free to establish their own laboratory-developed (home brew) system for mycobacterial sequencing. Molecular determination of drug resistance by sequence analysis has been successfully used for Mycobacterium tuberculosis and, in a limited way, for other mycobacteria. Drug resistance in M. tuberculosis is moderated by mutations in a handful of genes. Direct sequencing remains a challenge due to the paucity of mycobacteria in many specimen types especially in comparison with the amount of other bacteria present in complex matrices like sputum.

The year 1973 marked the opening of the Bosphorus Bridge, the first bridge to link the European and Anatolian sides of the city of Istanbul, the only city in the world that spans two continents. The same year, the first Istanbul Festival took place from 21 June to 15 July to help celebrate the fiftieth anniversary of the founding of the Turkish Republic. The festival included Western orchestral, opera, and chamber music, dance, theatre, and Turkish music. The festival was the idea of businessman Dr. Nejat Eczacibaşi, who gathered thirteen other Turkish businessmen to form the Istanbul Foundation for Culture and Arts (İstanbul Kültür ve Sanat Vakfi), a non-profit, non-governmental organization, abbreviated İKSV.

The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. In vitro, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated ex vivo and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. In vivo, single-agent INCB053914 inhibited Bcl-2-associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.

The SLOMYCO Sensititre panel and the custom JustOne strip (both from TREK Diagnostic Systems, Cleveland, OH) were evaluated for susceptibility testing of Mycobacterium avium complex isolates against clarithromycin. Seventy-one archived and prospectively collected isolates were tested using both the SLOMYCO panel and the JustOne strip, and the results were compared to those obtained using the BACTEC 460 (BD, Sparks, MD) radiometric method and a broth microdilution reference method. Results obtained by the SLOMYCO panel and the JustOne strip agreed with the BACTEC 460 method for 64/71 isolates (90%). Similarly, concordance with the broth microdilution method was 40/43 isolates (93%) for both test systems. The effect of the source medium on inoculum preparation was evaluated, and there were no differences noted in MICs, regardless of whether the inoculum was prepared from isolates grown in Middlebrook 7H9 medium, on Middlebrook 7H10 agar, or in VersaTREK broth culture bottles (Trek Diagnostics). Clarithromycin susceptibility testing of MAC using the SLOMYCO panel and the JustOne strip methods is easy to set up and simple to read and is readily incorporate into the clinical laboratory. These systems offer advantages over the BACTEC 460 system including the lack of a need for radioactive substrates, sharps, or costly instrumentation.

Several bromide salts composed of methylimidazolium cations possessing unsaturated sidechains (allyl-, 3-butenyl-, propargyl-, 2-butynyl-, and 2-pentynyl-) have been synthesized and characterized by multinuclear NMR, vibrational spectroscopy, and DSC, X-ray and elemental analysis. X-ray structures of 1-(2-butynyl)-3-methylimidazolium bromide, 1-propargyl-3-methylimidazolium bromide as well as the X-ray structure of 1-allyl-3- methylimidazolium bromide which was previously identified as a room temperature ionic liquid, were all determined.

The performance of the new VITEK 2 Advanced Colorimetry yeast identification (YST) card for use with the VITEK 2 system (bioMérieux, Inc., Hazelwood, MO) was compared to that of the API 20C AUX (API) system (bioMérieux SA, Marcy-l'Etoile, France) in a multicenter evaluation. A total of 12 quality control, 64 challenge, and 623 clinical yeast isolates were used in the study. Comparisons of species identification, platform reliability, and substrate reproducibility were made between YST and API, with API considered the reference standard. Quality control testing to assess system and substrate reproducibility matched expected results ≥95% of the time. The YST card correctly identified 100% of the challenge strains, which covered the species range of the manufacturer's performance claims. Using clinical isolates, the YST card correctly identified 98.5%, with 1.0% of isolates incorrectly identified and 0.5% unidentified. Among clinical isolates, the YST card generated fewer low-discrimination results (18.9%) than did API (30.0%). The time to identification with YST was 18 h, compared to 48 to 72 h with API. The colorimetric YST card used with the VITEK 2 provides a highly automated, objective yeast identification method with excellent performance and reproducibility. We found this system useful for timely and accurate identification of significant yeast species in the clinical microbiology laboratory.

A new class of energetic ionic liquids based upon 1-amino-3-alkyl-1,2,3-triazolium nitrates (alkyl = methyl, ethyl, n-propyl, 2-propenyl, and n-butyl) has been synthesized and characterized by vibrational spectra, multinuclear NMR, elemental as well as contaminant analyses, and DSC studies. A single crystal X-ray study was carried out for 1-amino-3-methyl-1,2,3-triazolium nitrate and the details will be presented.

The synthesis, characterization, theoretical calculations, and safety studies of energetic salts based on 1,2-bis(oxyamino)ethane, (H2NOCH2CH2ONH2), were carried out. The salts were characterized by vibrational (infrared, Raman), multinuclear NMR studies (1H, 13C), differential scanning calorimetry (DSC), elemental analysis, and initial safety testing (impact and friction sensitivity). Single crystal X-ray diffraction studies were carried out on the mono-perchlorate and the double nitrate salts, revealing the expected structures.

The identification of mycobacteria, other than the Mycobacterium tuberculosis complex, is a challenge for most routine clinical microbiology laboratories. Laboratories may use nucleic acid probe identification of Mycobacterium avium-M. intracellulare, Mycobacterium gordonae, and Mycobacterium kansasii, if financial resources are available. Currently, 109 species of mycobacteria have been described using nucleic acid sequencing. Phenotypic characters are generally incomplete in the literature, and for this reason, it is recommended that routine clinical microbiology laboratories refer cultures to state health laboratories or other reference laboratories for identification.

Reactions using the high nitrogen heterocycle 3,4,5-triamino-1,2,4-triazole (guanazine) with strong acids (HN03, HCl04, and "HN(N02)2") resulted in a family of highly stable salts. All of the salts were characterized using spectroscopic as well as single crystal x-ray diffraction studies. The x-ray structures are compared to that obtained from theoretical calculations (MP2/6-311+G(d,p) level). Initial safety testing (impact, friction) was carried out on all of the new materials.

The synthesis and characterization of 1 ,5-diamino- 1,2,3 ,4-tetrazolium perchlorate were carried out. Experimental evidence strongly supports the protonation of a nitrogen atom of the tetrazole ring, including the structure observed in a single crystal x-ray diffraction study of the title compound. Quantum chemical calculations were performed at the CCSD(T)/6-311G(2df,p)//MP2/6-311G(d,p) level of theory to determine the relative energies of all possible N-protonated structures of the 1,5.-diamino- 1,2,3,4-tetrazole ring. The predicted geometry of the most stable isomer compares favorably with the experimentally observed structure.

Recent molecular studies have shown Mycobacterium porcinum , recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776 T ) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776 T . Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, d -mannitol, i- myo -inositol, and catalase at 68°C. They were negative for l -rhamnose and d -glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776 T gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant d -sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.

The isolation and identification of a novel, slow-growing, scotochromogenic, mycobacterial species is reported. A strain, designated MUP 1182T, was isolated from a cervical lymph node of a 3-year-old child. MUP 1182T is alcohol- and acid-fast, with a lipid pattern that is consistent with those of species that belong to the genus Mycobacterium. It grows slowly at 25–37 °C, but does not grow at 42 °C. The isolate was revealed to be biochemically distinct from previously described mycobacterial species: it has urease and Tween hydrolysis activities and lacks nitrate reductase, 3-day arylsulfatase and β-glucosidase activities. Comparative 16S rDNA sequencing showed that isolate MUP 1182T represents a novel, slow-growing species that is related closely to Mycobacterium lentiflavum and Mycobacterium simiae. On the basis of these findings, the name Mycobacterium parmense sp. nov. is proposed, with MUP 1182T (=CIP 107385T=DSM 44553T) as the type strain.

The synthesis of three new families of heterocyclic-based salts was undertaken and accomplished. Three triazole systems, 1H-1,2,4-triazole, 4-amino-1,2,4-triazole, and 1H-1,2,3-triazole, were used as proton bases with nitric (HNO3), perchloric (HClO4), and dinitramidic (HN(NO2)2) acid systems. In all cases, stable salts were recovered and fully characterized by vibrational spectra (IR, Raman), multinuclear NMR spectroscopy, material balance, density measurements, and elemental analyses, as well as DSC, TGA and initial safety testing (impact). Many of these salts have melting points well below 100 °C, yet high decomposition onsets, defining them as new, highly energetic members of the well known class of materials identified as ionic liquids. Additionally, the single crystal X-ray diffraction study of 1,2,4-triazolium perchlorate was investigated, revealing the expected structure.

An evaluation of the MicroSeq 500 microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq 500 microbial identification system was accomplished with 59 American Type Culture Collection (ATCC) strains and 328 clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq 500 microbial identification system. Nucleic acid sequencing identified 58 of 59 (98.3%) ATCC strains to the species level or to the correct group or complex level. The identification results for 219 of 243 clinical isolates (90.1%) with a distance score of 1%; 35 (41.1%) were identified to the appropriate species level or group or complex level; 13 (15.3%) were identified to the species level. All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq 500 microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods. The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.

Six clinical isolates of the nonpigmented, rapidly growing species Mycobacterium mageritense were recovered from sputum, bronchial wash, blood, sinus drainage, and two surgical wound infections from separate patients in Texas, New York, Louisiana, and Florida. The isolates matched the ATCC type strain by PCR restriction enzyme analysis of the 65-kDa hsp gene sequence of Telenti, high-performance liquid chromatography, biochemical reactions, and partial 16S rRNA gene sequencing. These are the first isolates of this species to be described in the United States and the first isolates to be associated with clinical disease. Susceptibility testing of all known isolates of the species revealed all isolates to be susceptible or intermediate to amikacin, cefoxitin, imipenem, and the fluoroquinolones and sulfonamides but resistant to clarithromycin. Because of their phenotypic and clinical similarity to isolates of the Mycobacterium fortuitum third biovariant complex (sorbitol positive), isolates of M. mageritense are likely to go undetected unless selected carbohydrate utilization or molecular identification methods are used.

The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in “double-hit” DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL. Citation Format: Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 143. doi:10.1158/1538-7445.AM2017-143

The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumor immune response or to inhibit key proteins in the immunosuppressive tumor milieu. PD-1/PD-L1 axis blockade, CTLA-4 blockade and IDO1 inhibition are examples of the latter approach and have been utilized to reverse the suppressive tumor microenvironment, resulting in clinical benefit for cancer patients. Recent clinical and preclinical data have also demonstrated that combining these approaches results in enhanced therapeutic benefit. Notably, the IDO1-selective inhibitor epacadostat has been shown to increase the efficacy of two checkpoint inhibitors, the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody pembrolizumab, in patients with melanoma. Because both checkpoint receptors and IDO1 serve as negative regulators of the immune response, we also explored the ability of IDO1 inhibition to combine with agents that directly activate T cells through costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily. Rodent active surrogate agonist antibodies to 4-1BB, OX40 and GITR were tested with epacadostat in multiple preclinical models. In the B16-SIY melanoma model that does not express IDO1 in tumor cells, both epacadostat and anti-OX40 had little effect, but the combination resulted in enhanced efficacy. This was associated with increased infiltrates of CD8+ T cells and decreased numbers of FoxP3+ TILs. Increased numbers of SIY-reactive T cells were found in both the tumor and the TDLN post-therapy. In contrast, epacadostat did not provide any enhancement to the activity seen with 4-1BB. Clear combinatorial effects were seen with anti-GITR and epacadostat in the more inflamed, IDO1-expressing PAN02 pancreatic cancer model. These data suggest that IDO1 inhibition can be effective in combination with agents that agonize T cell costimulatory receptors as well as with agents that block coinhibitory receptors. Citation Format: Holly K. Koblish, Brendan Horton, Michael Hansbury, Sybil O'Connor, Kerri Lasky, Christina Stevens, Thomas Condamine, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Horacio Nastri, Gregory Hollis, Reid Huber, Thomas Gajewski, Peggy Scherle. Agonist antibodies targeting OX40 and GITR enhance the activity of the IDO1-selective inhibitor epacadostat in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2017-2618

Immune checkpoint blockade has shown considerable therapeutic promise in the clinic. However, single agent activity is compromised by the presence of suppressive myeloid cells, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAM) and polymorphonuclear (PMN) cells, in the tumor microenvironment. Epigenetic alterations can significantly contribute to the development of the immunosuppressive tumor microenvironment and recent data have suggested that combining epigenetic-based therapies with immunotherapeutic agents can lead to improved efficacy in preclinical models. Since Lysine Specific Demethylase 1 (LSD1) has been shown to play a critical role in hematopoiesis, we hypothesized that inhibition of LSD1 could have a direct effect on myeloid cell differentiation and potentially restore normal myelopoiesis in cancer patients. To test this hypothesis, we evaluated INCB059872, a potent, selective and orally available FAD-directed covalent inhibitor of LSD1 in several experimental models. In an in vitro differentiation assay, the majority of CD34+ progenitor cells were driven to a monocytic phenotype in the presence of INCB059872, while control treated cells differentiated toward granulocytic PMN cells. Similar results were observed in vivo. Using the orthotopic 4T1 mammary cancer model, the myeloid compartment was characterized in tumor tissues following treatment with INCB059872. Notably, the population of PMN-MDSC was significantly decreased in tumor tissues following oral administration of INCB059872, whereas the macrophage population was increased. These data suggest that INCB059872 can redirect myeloid differentiation toward monocyte/macrophages and inhibit the differentiation of PMN-MDSC in this syngeneic tumor microenvironment. Consistently, intratumoral T lymphocyte infiltration was increased following INCB059872 treatment. The combination of INCB059872 and α-PD-L1 antibody enhanced anti-tumor efficacy in the 4T1 orthotopic tumor model. Collectively, these data suggest that inhibition of LSD1 with INCB059872 can directly affect myeloid differentiation to reduce the accumulation of myeloid suppressive cells, restoring the tumor microenvironment to be more responsive to PD-1/PD-L1 axis blockade. This study supports the therapeutic potential for the combination of an LSD1 inhibitor with immuno-therapeutic agents to improve overall clinical response in cancer patients. Citation Format: Thomas Condamine, Steve Wang, Melody Diamond, Leslie Hall, Huiqing Liu, Antony Chadderton, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle, Holly Koblish, Sang Hyun Lee. The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2017-4635