Achilonu, Conrad Chibunna

Abstract Alveolar macrophages (AMs) are critical for host defense and immunity against Pneumocystis. Through surface-associated receptors, AMs recognize, phagocytose, and kill Pneumocystis organisms, producing cytokines that promote pathogen clearance but also contribute to lung inflammation and injury. The EphA2 receptor, a member of the receptor tyrosine kinase (RTK) family, contains an extracellular region that binds activating ligands (Ephrins) and, more recently, has been shown to bind fungal β-glucans, leading to activation of its intracellular tyrosine kinase domain. The EphA2 signaling pathway has important roles in cancer, angiogenesis, and inflammatory diseases such as atherosclerosis. EphA2 is highly expressed in epithelial and endothelial cells, and emerging evidence indicates that it serves as a critical regulator of host defense against bacterial, viral, and fungal pathogens. In oropharyngeal candidiasis, EphA2 was identified as a receptor for fungal β-glucans in oral epithelial cells, mediating proinflammatory signaling through signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. Our laboratory recently demonstrated that EphA2 on lung epithelial cells binds Pneumocystis organisms, and that β-glucans from these organisms activate downstream signaling leading to IL-6 release. However, the role of EphA2 signaling in AM immune responses during Pneumocystis pneumonia (PCP) remains unknown. To address this, we examined EphA2-deficient (EphA2−/−) AMs stimulated with Pneumocystis β-glucans. Compared to wild-type AMs, EphA2−/− AMs exhibited significantly decreased production of proinflammatory cytokines, including IL-6 and TNF-α. In CD4-depleted mouse models of PCP, EphA2−/− lungs displayed markedly reduced levels of inflammatory cytokines and increased organism burdens, indicating impaired immune activation but preserved fungal persistence. Importantly, in CD4-depleted mice undergoing standard trimethoprim-sulfamethoxazole (TMP/SMX) therapy, concurrent administration of the EphA2 small-molecule inhibitor ALW-II-41-27 significantly reduced lung TNF-α responses without evidence of toxicity. These findings demonstrate that EphA2 receptor signaling plays a critical role in regulating macrophage-mediated inflammatory responses to Pneumocystis. While EphA2 deficiency dampens pulmonary inflammation, it also compromises fungal clearance, highlighting a delicate balance between host defense and immunopathology. Our data suggest that targeted modulation of EphA2 signaling during anti-Pneumocystis therapy may provide a novel adjunctive approach to reduce detrimental lung inflammation associated with PCP, potentially improving outcomes in affected patients. This abstract is funded by: NIH R01 HL-62150

Abstract Rationale Pneumocystis pneumonia (PJP) remains a major opportunistic infection in immunocompromised hosts, with limited therapeutic options. Previous studies identified a conserved cysteine residue (C353) in Pneumocystisphosphoglucomutase (PGM), analogous to that found in other pathogenic fungi but absent in mammalian PGMs, suggesting an exploitable antifungal target. We aimed to define the functional significance and therapeutic potential of targeting Pneumocystis PGM. Methods Full-length P. jirovecii and P. murina PGM cDNAs were cloned into S. cerevisiae pgm2Δ yeast (deficient in PGM activity) using the pYES2.1 TOPO vector for heterologous expression. Recombinant yeast lysates were tested for PGM activity in the presence of the thiol-reactive inhibitor ISFP10 (0-200 μM). Parallel assays were performed using recombinant human PGM lacking C353. Yeast growth inhibition in these strains in the presence of ISFP10 was also measured over 72 hours. For in vitro and ex vivo assays, P. murina organisms were treated with ISFP10, and viability was quantified via qPCR of the mitochondrial 16S ribosomal copy number. Precision-cut lung slice (PCLS) ex vivo cultures from a murine PCP model and in vivo CD4-depleted mice were used to assess ISFP10 efficacy and toxicity. Results Both P. jirovecii and P. murina PGMs showed potent, dose-dependent inhibition of enzymatic activity by ISFP10 up to 12.5 μM, with no inhibition of human PGM at concentrations up to 200 μM. Yeast complementation assays confirmed that ISFP10 significantly reduced the growth of pgm2Δ yeast expressing Pneumocystis spp. PGM. In vitro incubation of P. murina with ISFP10 resulted in significant reductions in organismal 16S mitochondrial copy number, comparable to pentamidine. In ex vivo PCLS models, ISFP10 decreased P. murina burden without affecting uninfected tissue viability. In vivo, ISFP10 administration for seven days significantly reduced fungal burden in CD4-depleted mice with an acceptable safety profile. Conclusions These findings demonstrate that Pneumocystis PGM activity depends on a conserved cysteine residue that is selectively targetable by ISFP10. Pharmacologic inhibition of PGM impairs P. murina viability in vitro, ex vivo, and in vivo with minimal host toxicity. Collectively, these data establish PGM as a promising, fungus-specific antifungal target and support the further development of small-molecule inhibitors targeting Pneumocystis PGM. This abstract is funded by: NIH R01 HL-62150, R21 AI-181542

Abstract Background Alveolar macrophages (AMs) are central to host defense against Pneumocystis, mediating organism clearance while also contributing to lung inflammation. EphA2, a transmembrane receptor that binds fungal β-glucans, has emerged as a regulator of host–pathogen interactions, but its role in AM responses during Pneumocystis pneumonia (PCP) is unknown. Methods We evaluated AM inflammatory responses to Pneumocystis β-glucans in the presence and absence of EphA2. In vivo studies used a CD4-depleted mouse model of PCP comparing wild-type and EphA2-deficient (EphA2−/−) mice. Lung cytokine responses and organism burden were assessed. Additionally, EphA2 signaling was pharmacologically inhibited using ALW-II-41-27 during trimethoprim-sulfamethoxazole treatment. Results EphA2 deficiency significantly reduced AM proinflammatory cytokine responses to Pneumocystis β-glucans. In CD4-depleted PCP mice, EphA2−/− animals demonstrated decreased lung inflammatory cytokines, but increased organism burden compared to controls. Pharmacologic inhibition of EphA2 during antimicrobial therapy significantly suppressed lung inflammation. Conclusions EphA2 signaling plays a critical role in AM-mediated host defense against Pneumocystis by promoting inflammatory responses necessary for organism control. However, this pathway also contributes to lung inflammation. Targeting EphA2 may represent a novel strategy to modulate inflammation during severe PCP while preserving antimicrobial defense.

Introduction. Aspergillus fumigatus phosphoglucomutase ( Af PGM) is a central enzyme in fungal carbohydrate metabolism that catalyses the interconversion of glucose-1-phosphate and glucose-6-phosphate, generating precursors such as uridine diphosphate glucose required for the synthesis of key cell wall polysaccharides. Hypothesis/Gap Statement. Although Af PGM has been implicated in fungal viability and cell wall integrity, further functional and molecular characterization is required to better define its role in cell wall-associated processes and to evaluate its potential as a selective antifungal target. Aim. This study aimed to further characterize the functional contribution of Af PGM to fungal growth, cell wall-related phenotypes, biofilm formation and enzymatic activity. Methodology. A conditional A. fumigatus P alcA ::pgm mutant was used to assess growth, soluble β -glucan levels and biofilm formation. Functional complementation was evaluated by heterologous expression of Afpgm in a Saccharomyces cerevisiae pgm2Δ strain. Enzymatic inhibition was examined using the isothiazolone compound ISFP10. Results. Reduced Af PGM activity was associated with impaired growth, altered cell wall-related phenotypes, decreased soluble β -glucan levels and diminished biofilm biomass. Expression of Afpgm restored growth and sedimentation defects in the yeast pgm2Δ mutant to WT levels, confirming functional conservation. ISFP10 inhibited Af PGM activity and disrupted associated fungal phenotypes. Conclusion. These findings further define the functional role of Af PGM in fungal carbohydrate metabolism and cell wall-associated biology. The selective inhibition of Af PGM relative to human phosphoglucomutase supports its potential as a promising target for the development of novel antifungal therapies.

Abstract Candida auris is an emerging fungal pathogen with high rates of multidrug resistance, posing a significant global health threat. Immunosuppressed patients are particularly vulnerable to multidrug-resistant (MDR) C. auris infections. This systematic review and meta-analysis (SR-MA) aimed to assess the global prevalence of MDR-C. auris isolates in humans and their resistance to antifungal drugs, primarily fluconazole (FLZ), voriconazole (VOR), and amphotericin B (AmB). A comprehensive search was conducted in PubMed, Scopus, and ScienceDirect databases on published studies regarding clinical C. auris isolates and the use of antifungal susceptibility testing assay from 17 March 2015 to 1 February 2024. Meta-analysis was conducted using random-effects models to determine the estimated pooled prevalence of MDR-C. auris isolates and their antifungal resistance profiles from immunosuppressed humans. A total of 81 research studies were included in the final analysis, with the overall pooled prevalence of the three most important antifungal drugs; FLZ (92.5% | 95% CI: 89.5–94.7), VOR (49.0% | 95% CI: 37.6–60.4), and AmB (51.0% | 95% CI: 42.3–59.7). According to the subgroup analyses, FLZ-resistant C. auris was the most prevalent isolate observed in the five continents reporting compared to the VOR- and AmB-resistant C. auris having lower pooled prevalence. Further, higher pooled prevalence of MDR-C. auris isolates were observed from males (68.2%, n = 578) compared to females (37.1%, n = 283). Finally, the global pooled prevalence for mortality in immunosuppressed patients with MDR-C. auris from 13 research studies was 41.5% (95% CI: 29.9–54.2). This SR-MA study establishes the variation of MDR-C. auris resistance patterns observed from different continents and the importance to conduct further research studies using potent antifungal drugs. Accordingly, it is imperative to establish stringent surveillance on the increasing prevalence of antifungal resistance particularly in low-income regions with limited research resources to prevent increasing prevalence of MDR-C. auris.

Primary tumor resection and liver transplantation are the only curative treatment options for the management of cholangiocarcinoma (CCA). However, for patients with advanced or metastatic disease, palliative systemic therapy remains the only treatment option. The development of targeted therapeutics has begun to shift the treatment paradigm in CCA. Targets of interest in CCA include mutated isocitrate dehydrogenase-1 (mIDH-1), human epidermal growth factor receptor 2 (HER2) overexpression/amplification, and fibroblast growth factor receptor 2 (FGFR2) fusion, in addition to less frequently observed targets such as BRAF V600E, deficient mismatch repair/high microsatellite instability (dMMR/MSI-H), and high tumor mutation burden (TMB-H). These targets are observed in varying frequency among patients with intrahepatic CCA and extrahepatic CCA. Multiple novel therapies have been developed to exploit each of these targets, with some having received United States Food and Drug Administration approval for use in the second-line setting. In the current review, we discuss targets of interest in CCA and summarize current evidence evaluating available therapies directed at these targets.

Although uncommon, extrahepatic cholangiocarcinoma (EHCC) is a deadly malignancy, and the treatment approaches remain controversial. While surgery remains the only cure, few patients are candidates for resection up front, and there are high rates of both local and distant failure following resection. Herein, we systematically review the available evidence regarding treatment approaches for patients with EHCC, including surgery, radiation, and chemotherapy. The evidence regarding treatment outcomes was assessed using the Population, Intervention, Comparator, Outcome, and Study design (PICOS) framework. A summary of recommendations based on the available literature is outlined for specific clinical scenarios encountered by providers in the clinic to guide the management of these patients.

Biliary tract cancers are a diverse and aggressive malignancy that carry a poor chance for curative treatment and significant associated mortality. Current first-line treatment only extends median overall survival to roughly 1 year and is associated with a significant adverse event profile. Recently, advancements in genetic sequencing have opened new avenues of targeted treatment. In cholangiocarcinoma, FGFR2 alterations have been shown to be present in roughly 10–15% of intrahepatic cholangiocarcinoma. Pemigatinib, a FGFR1–4 inhibitor, has been shown to significantly extend survival in the second-line setting to over 20 months in patients who harbor FGFR2 fusions. Here, we outline the development and future direction of pemigatinib and other FGFR2 inhibitors in the field of advanced biliary tract cancers.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies originating in the biliary tree, and associated with poor prognosis. Until recently, treatment options have been limited to surgical resection, liver-directed therapies, and chemotherapy. Identification of actionable genomic alterations with biomarker testing has revolutionized the treatment paradigm for these patients. However, several challenges exist to the seamless adoption of precision medicine in patients with CCA, relating to a lack of awareness of the importance of biomarker testing, hurdles in tissue acquisition, and ineffective collaboration among the multidisciplinary team (MDT). To identify gaps in standard practices and define best practices, multidisciplinary hepatobiliary teams from the University of California (UC) Davis and UC Irvine were convened; discussions of the meeting, including optimal approaches to tissue acquisition for diagnosis and biomarker testing, communication among academic and community healthcare teams, and physician education regarding biomarker testing, are summarized in this review.

Biliary tract cancers (BTCs) are a heterogeneous group of adenocarcinomas that originate from the epithelial lining of the biliary tree. BTCs are characterized by presentation with advanced disease precluding curative surgery, rising global incidence, and a poor prognosis. Chemotherapy is the mainstay of the current treatment, which results in a median overall survival of less than one year, underscoring the need for novel therapeutic agents and strategies. Next-generation sequencing-based molecular profiling has shed light on the underpinnings of the complex pathophysiology of BTC and has uncovered numerous actionable targets, leading to the discovery of new therapies tailored to the molecular targets. Therapies targeting fibroblast growth factor receptor (FGFR) fusion, isocitrate dehydrogenase (IDH) mutations, the human epidermal growth factor receptor (HER) family, DNA damage repair (DDR) pathways, and BRAF mutations have produced early encouraging results in selected patients. Current clinical trials evaluating targeted therapies, as monotherapies and in combination with other agents, are paving the way for novel treatment options. Genomic profiling of cell-free circulating tumor DNA that can assist in the identification of an actionable target is another exciting area of development. In this review, we provide a contemporaneous appraisal of the evolving targeted therapies and the ongoing clinical trials that will likely transform the therapeutic paradigm of BTC.