Mukil Natarajan

Autoimmune Regulator Deficiency Results in a Decrease in STAT1 Levels in Human Monocytes

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by biallelic autoimmune regulator (AIRE) mutations that manifests with chronic mucocutaneous candidiasis (CMC) and autoimmunity. Patients with STAT1 gain-of-function (GOF) mutations also develop CMC and autoimmunity; they exhibit increased STAT1 protein levels at baseline and STAT1 phosphorylation (pSTAT1) upon interferon (IFN)-γ stimulation relative to healthy controls. AIRE interacts functionally with a protein that directly regulates STAT1, namely protein inhibitor of activated STAT1, which inhibits STAT1 activation. Given the common clinical features between patients with AIRE and STAT1 GOF mutations, we sought to determine whether APECED patients also exhibit increased levels of STAT1 protein and phosphorylation in CD14+ monocytes. We obtained peripheral blood mononuclear cells from 8 APECED patients and 13 healthy controls and assessed the levels of STAT1 protein and STAT1 tyrosine phosphorylation at rest and following IFN-γ stimulation, as well as the levels of STAT1 mRNA. The mean STAT1 protein levels in CD14+ monocytes exhibited a ~20% significant decrease in APECED patients both at rest and after IFN-γ stimulation relative to that of healthy donors. Similarly, the mean peak value of IFN-γ-induced pSTAT1 level was ~20% significantly lower in APECED patients compared to that in healthy controls. The decrease in STAT1 and peak pSTAT1 in APECED patients was not accompanied by decreased STAT1 mRNA or anti-IFN-γ autoantibodies; instead, it correlated with the presence of autoantibodies to type I IFN and decreased AIRE−/− monocyte surface expression of IFN-γ receptor 2. Our data show that, in contrast to patients with STAT1 GOF mutations, APECED patients show a moderate but consistent and significant decrease in total STAT1 protein levels, associated with lower peak levels of pSTAT1 molecules after IFN-γ stimulation.

GM-CSF Therapy in Human CARD9 Deficiency

CARD9 deficiency is associated with life-threatening fungal infections of the CNS. This work shows that the H-RAS/RASGRF1/ERK pathway is intact in the c.170G>A (p.R57H) CARD9 missense mutation, a finding that correlates with the clinical outcome of GM-CSF therapy.

VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans

Background Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. Objectives To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. Methods MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. Results Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. Conclusions VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.

Aspergillosis, eosinophilic esophagitis, and allergic rhinitis in signal transducer and activator of transcription 3 haploinsufficiency

STAT3 haploinsufficiency caused by a novel STAT3 splice site mutation is associated with elevated IgE, allergic rhinitis, eosinophilic esophagitis, and invasive aspergillosis. This case expands our understanding of the spectrum of disease associated with STAT3 mutations.

Pulmonary Histoplasma Infection After Allogeneic Hematopoietic Stem Cell Transplantation: Case Report and Review of the Literature

Abstract Histoplasmosis causes a wide spectrum of clinical illness, including disseminated infection in the immunocompromised. We report a case of pulmonary histoplasmosis in an allogeneic stem cell transplant recipient and review the literature on this topic. Histoplasmosis in this patient population is uncommon, but it is associated with poor outcome.

Developing a comprehensive system for content-based retrieval of image and text data from a national survey

The article describes the status of our ongoing R&D at the U.S. National Library of Medicine (NLM) towards the development of an advanced multimedia database biomedical information system that supports content-based image retrieval (CBIR). NLM maintains a collection of 17,000 digitized spinal X-rays along with text survey data from the Second National Health and Nutritional Examination Survey (NHANES II). These data serve as a rich data source for epidemiologists and researchers of osteoarthritis and musculoskeletal diseases. It is currently possible to access these through text keyword queries using our Web-based Medical Information Retrieval System (WebMIRS). CBIR methods developed specifically for biomedical images could offer direct visual searching of these images by means of example image or user sketch. We are building a system which supports hybrid queries that have text and image-content components. R&D goals include developing algorithms for robust image segmentation for localizing and identifying relevant anatomy, labeling the segmented anatomy based on its pathology, developing suitable indexing and similarity matching methods for images and image features, and associating the survey text information for query and retrieval along with the image data. Some highlights of the system developed in MATLAB and Java are: use of a networked or local centralized database for text and image data; flexibility to incorporate new research work; provides a means to control access to system components under development; and use of XML for structured reporting. The article details the design, features, and algorithms in this third revision of this prototype system, CBIR3.

Acute Epiglottitis in the Immunocompromised Host: Case Report and Review of the Literature

Abstract We present a case of acute epiglottitis in a 16-year-old with severe aplastic anemia. He was admitted with a history suggestive of a severe upper airway infection and an absolute neutrophil count of 0 per cubic millimeter. Despite his immunocompromised state, he presented with the classical signs and symptoms of epiglottitis. We review here the presentation and comorbidities of immunocompromised patients with epiglottitis. In addition, the appropriate choice of empirical antibiotic therapy is important for the management of epiglottitis in immunocompromised patients, especially in the post–Haemophilus influenza type B vaccination era. In our patient, Enterobacter cloacae was isolated from endoscopically directed throat cultures, and treatment was successful without the need for intubation. The current literature suggests that in immunocompromised patients, particularly those who are neutropenic, there is a potentially wide range of organisms, both bacterial and fungal, that may play a role in the pathology of acute epiglottitis.

VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice

Background Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.

De novo STAT3 Mutation in a Patient with Fatal, Treatment-Refractory Sino-orbital Aspergillosis

Abstract Background Signal transducer and activator of transcription 3 (STAT3) loss-of-function mutations in humans result in Job’s syndrome, characterized by elevated IgE, bacterial infections, chronic mucocutaneous candidiasis, pulmonary aspergillosis due to structural lung disease, and connective tissue abnormalities. Methods Whole exome sequencing (WES) was performed on a putatively immunocompetent patient with sino-orbital aspergillosis and his parents. Evaluation of STAT3 phosphorylation (pSTAT3) was performed by flow cytometry on peripheral blood mononuclear cells after IL-6 stimulation. Results A 37-year-old-male with a history of eosinophilic esophagitis developed a locally invasive sino-orbital infection with progressive cavernous sinus involvement associated with facial numbness, diplopia, and visual loss. Biopsy showed chronic inflammation and invasive fungal hyphae. Cultures grew an isolate of Aspergillus fumisynnematus that was identified by morphology and the sequences of β-tubulin and Mcm7. MICs (in µg/ml) for the isolate were: micafungin <=0.015, amphotericin 2, posaconazole 0.25, terbinafine 0.06, and voriconazole 0.25. The patient underwent multiple surgical debridements and was treated over time with various antifungals (amphotericin B, micafungin, terbinafine, voriconazole, posaconazole), adjunct cytokines (IFN-γ, GM-CSF), and hyperbaric oxygen. However, the infection progressed into the right middle cranial fossa and meninges and the patient died 1 year after symptoms began. WES revealed a de novo splice-site mutation in STAT3 (c.1140-3C>G). cDNA sequencing showed nonsense mediated decay of the affected allele. No mutations in CARD9 or NADPH oxidase subunits were found; a DHR test was normal. The patient had normal blood myeloid cell subsets. Serum IgE level was elevated at 833 IU/ml. After stimulation with IL-6, the patient’s memory CD4+ T cells and CD11c+ myeloid cells had reduced pSTAT3 levels compared with control cells. Cellular analysis of SOCS3, a STAT3-dependent downstream target, is underway to evaluate for functional STAT3 haploinsufficiency. Conclusion A novel de novo STAT3 splice site mutation results in impaired pSTAT3, and is associated with elevated IgE, eosinophilic esophagitis, and sino-orbital aspergillosis without other common features of Job’s syndrome Disclosures All authors: No reported disclosures.

VT-1598 Inhibits the in vitro Growth of Mucosal Candida Isolates and Protects Against Oropharyngeal Candidiasis in IL-17 Deficient Mice

Abstract Background Patients with chronic mucocutaneous candidiasis (CMC) often develop azole-resistant Candida infections, making treatment difficult due to lack of oral antifungal drug options. VT-1598 is a novel broad-spectrum fungal CYP51 inhibitor designed for exquisite selectivity for the fungal target versus human CYP enzymes to circumvent classic azole side effects like drug–drug interactions. We report the efficacy of VT-1598 in the treatment of oral Candida infection (including by azole-resistant strains). Methods The in vitro MIC values of 28 Candida species isolated from patients with CMC due to AIRE mutations were tested against VT-1598 and fluconazole (FLC), using CLSI broth microdilution M27-S4. Plasma VT-1598 levels were measured using LC–MS/MS with electrospray ionization. Tongue fungal load was determined in IL-17 deficient Act1- /- mice following sublingual C. albicans infection and once-daily oral treatment for 4 days with 25 mg/kg FLC or 3.2, 8, and 20 mg/kg VT-1598 starting 18 hours post-infection. Results Among 28 Candida isolates tested (22 C. albicans, three C. glabrata, and one each of C. utilis, C. dubiliensis, and C. krusei), 10 (36%) were not susceptible to FLC, based on CLSI breakpoints (>4 mg/ml). Remarkably, all 28 isolates were highly susceptible to VT-1598 (MIC50 and MIC90, 0.06 and 0.125 mg/ml, respectively). Oral administration of VT-1598 led to mean drug levels in mouse plasma (2.0, 3.0, and 11 mg/ml at the low, mid, and high doses, respectively) that were higher than the MIC values. In vivo, VT-1598 was significantly more effective, compared with FLC, against FLC-susceptible and -resistant C. albicans, and led to elimination of fungal growth even at the lowest tested dose (3.2 mg/kg). After a 10-day washout period from the last dose, mice treated with VT-1598 did not have mucosal fungal growth, while mice treated with FLC had tongue fungal loads similar to vehicle control. Conclusion VT-1598 shows in vitro activity against mucosally derived Candida, including FLC-resistant strains. In vivo, VT-1598 achieves high plasma concentrations and eliminates viable C. albicans, even at low doses and after an extended washout period. These data indicate that VT-1598 may be a significantly improved treatment option for patients with CMC. Disclosures C. M. Yates, Viamet Pharmaceuticals, Inc.: Employee, Salary. O. J. Cohen, Viamet Pharmaceuticals, Inc.: Employee, Salary. R. J. Schotzinger, Viamet Pharmaceuticals, Inc.: Employee, Salary. E. P. Garvey, Viamet Pharmaceuticals, Inc.: Employee, Salary. M. Lionakis, Viamet Pharmaceuticals: Research support (CRADA), Research support