Elevated opening pressures on lumbar puncture were followed in 7 nonimmunocompromised patients being treated for a postinfectious inflammatory response syndrome that had begun following antifungal treatment of cryptococcal meningitis. During the initial period of high-dose corticosteroid therapy, opening pressures on lumbar puncture fell an average of 14 cm. Despite this, 5 of 7 patients later required a ventriculoperitoneal shunt for pressure control.

Bloodstream infection with Candida species is associated with high mortality, yet the molecular basis of vascular adhesion and invasion remains unclear. We identify the endothelial surface mucin MUC16 as a key host factor enabling fungal vascular adherence. The MUC16 SEA domain directly engages the Candida adhesin Als3p and endothelial N-cadherin to form a ternary complex that promotes endothelial attachment and vascular invasion. Genetic deletion of Muc16 in mice markedly reduced fungal vascular binding, renal fungal burden, and Ly6Ghi CD11b+ neutrophil recruitment following intravenous infection, and diminished oral colonization in an oral model. Inflammatory induction of MUC16 occurred via MINCLE-, TLR9-, and Dectin-1-dependent signaling, converging on CARD9. In clinical cohorts, a MUC16 T10155I variant and elevated circulating MUC16 were associated with increased 30-day mortality. These findings establish MUC16 as a mechanistic link between endothelial sensing and Candida vascular invasion, with prognostic relevance in candidemia.

Anti-cytokine autoantibodies represent an expanding field in immunology, and their study has revealed crucial insights into immune cell dysfunction. These autoantibodies are now classified by the International Union of Immunological Societies (IUIS) as phenocopies of primary immunological deficiencies within the broader category of inborn immunity defects. Indeed, the critical importance of these autoantibodies became starkly apparent during the COVID-19 pandemic when patients with type I interferon autoantibodies showed significantly higher rates of severe illness. This review examines how neutralizing autoantibodies, exemplified by those targeting granulocyte monocyte stimulating factor, can compromise immune function in otherwise immunocompetent individuals, making them more susceptible to specific fungal and bacterial infections. This understanding highlights the crucial role of anti-cytokine antibodies in infection susceptibility and immune system regulation.

Therapies to reduce neuroinflammation following resolution of acute central nervous system (CNS) infections are urgently needed, particularly for patients with non-HIV-associated cryptococcal meningoencephalitis complicated by a postinfectious inflammatory response syndrome (cPIIRS). To identify druggable targets in cPIIRS, patient cerebral spinal fluid samples underwent transcriptional analysis, revealing a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway dominance in neuroinflammatory gene signatures. MurinecPIIRS models recapitulated this pathway predominance and treatment with the JAK inhibitor ruxolitinib, confirmed a mechanistic requirement for this pathway in disease pathology. Ruxolitinib treatment improved markers of neuronal damage, reduced activated T cell and myeloid cells, and improved weight. On the basis of these findings, we conducted a first-in-human ruxolitinib treatment of patients with cPIIRS (NCT00001352). Ruxolitinib treatment of six patients led to demonstrated tolerability, reductions in inflammatory biomarkers and activated immune cells, and improved brain imaging. These results advocate for pathway-instructed therapeutics in neuroinflammatory diseases and endorse JAK inhibitors in further clinical studies of cPIIRS.

Abstract Background Cryptococcal meningoencephalitis (CM) is a major cause of mortality in HIV/AIDS, transplant recipients and previously healthy individuals. In the latter, a post-infectious inflammatory response syndrome (PIIRS) has been noted to have a favorable response to corticosteroids although long-term responses have not been studied. Methods In addition to standard antifungal therapy, 15 patients admitted with CM/PIIRS from 2015-2020 were treated with standard pulse – taper corticosteroid therapy (PCT) consisting of intravenous methylprednisolone 1 gm daily for 1 week followed by oral prednisone 1 mg/kg/d, tapered based on clinical and radiological response. One patient was excluded from the study due to death from an unrelated cause within 6 months of diagnosis. Comprehensive neurocognitive testing and MRI brain scans were performed one year after diagnosis to assess clinical status. Results 80% of patients were male, median age 51 years. Median time from antifungal treatment to steroid initiation was 6 weeks. Eleven of 14 patients were neuropsychologically evaluated using performance-based neurocognitive tests. A one-sample T- test showed that the cohort obtained significantly lower scores in memory, learning, language, and information processing/psychomotor speed test in comparison to a normative sample. Also, the mean memory, learning, psychomotor, and information processing speed scores were in the low average range. These findings suggest very mild cognitive deficits in this cohort one year post diagnosis. With the exception of one patient whose MRI brain showed worsened findings at the one-year mark due to a neuro-inflammatory flare, radiological findings suggested stability or improvement for the remaining 13 patients. CSF cultures remained negative. Conclusion Neurocognitive testing performed in a small cohort of previously healthy patients with CM-PIIRS showed an absence of moderate or major neurocognitive deficits one year post diagnosis with minimal adverse effects and no reoccurrence of infection. Brain MRI findings were also stable or improved for the majority. Disclosures All Authors: No reported disclosures

Cryptococcus meningoencephalitis (CM) is a lethal fungal disease. Clinical observations and animal model studies document that unchecked inflammation in the brain contributes to the disease’s progression. Regulatory T cells (Tregs) are critical for maintaining immune balance, yet their precise role and mechanisms in preventing pathological neuroinflammation during CM, as well as their therapeutic potential, remain poorly understood. Our study reveals that Tregs are present in the CSF of CM patients and are accumulated in mouse brain over the course of disease progression; Treg depletion exacerbates Th1-driven brain inflammation, worsens neurological symptoms, and accelerates mortality, despite a reduction in fungal burden; Brain single-cell RNA-seq analysis shows high expression of CCR8 on Tregs and anti-CCR8 treatment significantly reduced Treg accumulation in the brain, revealing Treg recruitment in the brain depends on CCR8-CCL1 axis; Furthermore, brain-infiltrating Tregs suppress inflammation via the anti-inflammatory cytokine IL-10, while their production of Areg promotes IFN-γ production by Teffs, in part reducing Treg efficiency in suppressing CM inflammation. Finally, a promising Treg-enhancing immunotherapy using low-dose IL-2 treatment improves mice survival and neurological function significantly. Taken together, our results identify that CCR8-CCL1-recruited Tregs in the brain could serve as a potential therapeutic strategy to mitigate immunopathology during CM. Microbial, Parasitic, and Fungal Immunology (MPF)

Immunological studies of persons with cryptococcal disease without known immunocompromise could inform vaccine development and identify novel requirements for antifungal immunity. We tested recall responses to heat-killed (HK) Cryptococcus and recombinant cryptococcal candidate vaccine antigens in peripheral blood T cells from subjects with a history of cryptococcosis and no recognized predisposing condition at the time of study enrollment, alongside healthy controls. In response to stimulation with HK Cryptococcus, CD4+ and CD8+ T cells from the cryptococcosis subjects had greater activation compared to healthy controls. Responses to recombinant antigens, while less robust, were also more frequent in patient samples compared to healthy controls. Fungal strains stimulated much higher interferon gamma (IFNγ) secretion compared with interleukin (IL)-17A and IL-4. A mouse model of vaccination and cryptococcal infection was established to determine the degree to which antigen-specific IFNγ responses in the periphery reflect responses in the tissue. Peripherally, the mouse model mimicked the human studies with the additional finding that responses were greatest in mice following both vaccination and infection. Moreover, responses in splenocytes and lung cells exceeded those in PBMCs suggesting PBMC responses underestimate those at sites of infection. In summary, in otherwise immunocompetent patients with cryptococcosis, antigen-stimulated CD4+ and CD8+ T cell activation, as measured by cytokine, was intact and biased towards a T-helper 1 (Th1) response. The mouse data support vaccination as a strategy to boost immune responses to prevent clinical Cryptococcus infection.

The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus -specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[ 18 F]fluorocellobiose ([ 18 F]FCB) by enzymatic conversion of 2-deoxy-2-[ 18 F]fluoroglucose ([ 18 F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [ 18 F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus , bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of β-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [ 18 F]FCB into glucose and [ 18 F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [ 18 F]FCB is a promising and clinically translatable Aspergillus -specific PET tracer.

Follow-up of previously healthy patients surviving cryptococcal meningitis found that cryptococcal antigen could be detected for >1 year in serum from 38 of 44 (86%) patients and in cerebrospinal fluid (CSF) from 20 of 31 patients (67%), far beyond the time of culture conversion. The speed of titer decline, measured as the number of days for a 2-fold drop in titer to occur, was slower in serum than in CSF. The speed of decline of antigen titers was much slower in serum and CSF for patients infected with Cryptococcus gattii than Cryptococcus neoformans. The speed of decline in CSF and serum titers was also much slower in patients who had received a ventriculoperitoneal shunt for increased intracranial pressure. The variable and extraordinarily slow rate of clearance in our patients did not appear to reflect differences in disease control but rather differences in species and shunting for increased intracranial pressure.

Post-infection inflammatory syndromes have been increasingly recognized as a cause of host damage in a variety of infectious diseases including tuberculosis, bacterial meningitis, and COVID-19. Recently, a post-infectious inflammatory response syndrome (PIIRS) was described in non-HIV-infected cryptococcal fungal meningoencephalitis (CM) as a major cause of mortality. Inflammatory syndromes are particularly severe in neurological infections due to the skull's rigid structure which limits unchecked tissue expansion from inflammatory-induced edema. In the present studies, neurologic transcriptional pathway analysis utilizing a murine PIIRS model demonstrated a predominance of Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation. JAK/STAT inhibitor treatment resulted in improvements in CNS damage markers, reductions in intrathecal CD44hiCD62lo CD4+ effector CD4+ T-cells and MHC II+ inflammatory myeloid cells, and weight gains in mice, the latter after treatment with antifungals. Based on these data, pathway-driven steroid-sparing human treatment for steroid-refractory PIIRS was initiated using short courses of the JAK/STAT inhibitor ruxolitinib. These were well tolerated and reduced activated HLA-DR+ CD4+ and CD8+ cells and inflammatory monocytes as well as improved brain imaging. Together, these findings support the role of JAK/STAT in PIIRS as well as further study of JAK/STAT inhibitors as potential adjunctive therapy for PIRS and other neural inflammatory syndromes.

Abstract Recently, a PIIRS was described in non-HIV infected cryptococcal meningoencephalitis (CM) as a significant cause of neuropathology and mortality. Inflammatory syndromes are particularly severe in neurological infections due to the skull's rigid structure. In the present study, transcriptional profiling of RNA from brains of PIIRS model mice at peak inflammation identified a predominance of STAT1/STAT3-associated downstream genes. Since JAK is known to activate both pathways, we hypothesized that the JAK/STAT inhibitor ruxolitinib could reduce neuro-damaging inflammation from CM. Treatment with ruxolitinib reduced intrathecal STAT1/STAT3-associated gene expression as well as CD44hiCD62loCD4+ effector CD4+ T-cells and MHC II+ inflammatory myeloid cells with improved neuropathology and weight. Based on these data, the first human treatment of PIIRS with ruxolitinib was initiated in 5 patients with residual inflammation despite corticosteroids (CS). Ruxolitinib reduced cerebrospinal fluid (CSF) activated HLA-DR+ CD4+ and CD8+ cells, NK cells, and inflammatory monocytes as well as soluble markers sIL-2R, and IL-10 with a trend in IL-6 on a constant dose of CS. MRI brain imaging also demonstrated significant improvements and therapy was well tolerated. Together, these findings demonstrate a role for the JAK/STAT pathway in PIIRS and support ruxolitinib as a potential adjunctive therapy for further randomized clinical trials of PIIRS as a steroid-sparing therapeutic.

Background: A clearer understanding is needed about the use of brain MRI in non-HIV patients with cryptococcal meningitis. Methods: Cerebral CT and MRI were studied in 62 patients in a multicenter study of cryptococcal meningitis in non-HIV patients. CT was performed in 51 and MRI in 44. MRI results are reported for the images read at NIH for 29 of the 44 patients. CT reports obtained from the original REDCap database were added to calculate the incidence of normal findings. Results: CTs were read as normal in 24 of 51 (47%), MRIs were normal in 10% (three of 29). The most characteristic lesions of cryptococcal meningitis on MRI were small basal ganglia lesions representing dilated perivascular spaces in 24% and basal ganglia lesions with restricted diffusion (infarcts) in 38%. In the 18 patients who received contrast, contrast-enhancing lesions, likely representing masses of cryptococci and inflammatory cells, were found in the basal ganglia in 22% and elsewhere in the brain in 22%. Meningeal enhancement was seen in 56%, ependymal enhancement in 24%, and choroid plexus enhancement in 11%. Hydrocephalus was found in five (18%), though increased intacranial pressure was not detected. Suboptimal imaging (n = 6), lack of contrast administration (n = 11) and lack of follow-up, however, markedly limited the accurate assessment of abnormalities in multiple cases. Conclusion: MRI characteristics of non-HIV cryptococcal meningitis include hydrocephalus, meningeal and ependymal enhancement and basal ganglia lesions. Optimal imaging is, however, necessary to maximize the diagnostic and prognostic usefulness of MRI.

Dog breeds represent canine sub-populations with distinctive phenotypic features and limited genetic diversity. We have established a resource to study breed-specific genetic diversity. Utilising genetic resources within our laboratory biobank, public domain genotype data and the phylogenetic framework of 23 breed clades, the primary objective for this study was to identify genomic regions that differentiate the Bullmastiff breed. Through application of a composite index analysis (CSS), genomic signatures were identified in Bullmastiffs when compared to the formative breeds, Mastiffs and Bulldogs, and to 22 other breed groups. Significant regions were identified on 15 chromosomes, with the most differentiated regions found on CFA1, CFA9, and CFA18. These regions may reflect genetic drift following establishment of the breed or the effects of selective breeding during development of the modern Bullmastiff. This was supported by analysis of genes from the identified genomic regions, including 458 genes from the multi-clade analysis, which revealed enriched pathways that may be related to characteristic traits and distinct morphology of the breed. The study demonstrates the utility of the CSS method in breed-specific genome analysis and advances our understanding of genetic diversity in Bullmastiff dogs.

The chemistry of metal ions with human pathogens is essential for their survival, energy generation, redox signaling, and niche dominance. To regulate and manipulate the metal ions, various enzymes and metal chelators are present in pathogenic bacteria. Metalloenzymes incorporate transition metal such as iron, zinc, cobalt, and copper in their reaction centers to perform essential metabolic functions; however, iron and copper have gained more importance. Multicopper oxidases have the ability to perform redox reaction on phenolic substrates with the help of copper ions. They have been reported from Enterobacteriaceae, namely Salmonella enterica, Escherichia coli, and Yersinia enterocolitica, but their role in virulence is still poorly understood. Similarly, superoxide dismutases participate in reducing oxidative stress and allow the survival of pathogens. Their role in virulence and survival is well established in Salmonella typhimurium and Mycobacterium tuberculosis. Further, to ensure survival against stress, like metal starvation or metal toxicity, redox metalloenzymes and metal transportation systems of pathogens actively participate in metal homeostasis. Recently, the omics and protein structure biology studies have helped to predict new targets for regulation the colonization potential of the pathogenic strains. The current review is focused on the major roles of redox metalloenzymes, especially MCOs and SODs of human pathogenic bacteria.

Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.

Non-HIV cryptococcal meningoencephalitis (CM) in previously healthy individuals is often complicated by a post-infectious inflammatory response syndrome (c-PIIRS) characterized by neurologic deterioration after appropriate antifungal therapy with sterilization of CSF fungal cultures. c-PIIRS results from an excessive inflammatory response to fungal antigens released during fungal lysis, mediated by IFN-γ, IL-6, and activated T-helper cells, leading to immune-mediated host damage that responds to pulse-corticosteroid taper therapy (PCT). Typically, oral steroids may take up to a year to taper, and occasionally, patients will be refractory to steroid therapy or may demonstrate high-risk lesions such as those involving intracranial arteries. Also, patients can have problematic side effects from prolonged corticosteroids. Hence, appropriate adjunctive agents are needed to reduce corticosteroid doses in the treatment of c-PIIRS. Due to a possible role of IL-6 in pathogenesis, IL-6 receptor blockade by tocilizumab may be useful in the treatment of c-PIIRS. Two previously healthy patients with non-HIV cPIIRS were seen at the NIH. Due to concerns for intracranial vascular rupture in an area of inflammation (Patient 1) and intractable symptoms on high-dose oral corticosteroids (Patient 2) with evidence of persistent CSF inflammation, patients were treated with 4–8 mg/kg tocilizumab every 2 weeks while maintained on a constant dose of prednisone. Two patients exhibited rapid immunological improvement following treatment with tocilizumab. Patient 1 remained vascularly stable, and Patient 2 had near resolution of headaches with improvement in mental status as evidenced by improved MOCA score. The two had improved CSF inflammatory parameters and no significant side effects. Both CSF cultures remained negative throughout treatment. Tocilizumab may be a safe adjunctive treatment for CM-related PIIRS suggesting further study.

Lymphoma is the most common haematological malignancy affecting dogs and has a high incidence in the Bullmastiff breed. The aim of this study was to identify risk loci predisposing this breed to the disease. The average age of lymphoma diagnosis in 55 cases was less than 6 years, similar to the median age of 64 cases from our clinical and pathology databases. When fine-scale population structure was explored using NETVIEW, cases were distributed throughout an extended pedigree. When genotyped cases (n = 49) and dogs from the control group (n = 281) were compared in a genome-wide association analysis of lymphoma risk, the most prominent associated regions were detected on CFA13 and CFA33. The top SNPs in a 5.4 Mb region on CFA13 were significant at a chromosome-wide level, and the region was fine-mapped to ~1.2 Mb (CFA13: 25.2–26.4 Mb; CanFam3.1) with four potential functional candidates, including the MYC proto-oncogene bHLH transcription factor (MYC) and a region syntenic with the human and mouse lncRNA Pvt1 oncogene (PVT1). A 380 Kb associated region at CFA33: 7.7–8.1 Mb contained the coding sequence for SUMO specific peptidase7 (SENP7) and NFK inhibitor zeta (NFKBIZ) genes. These genes have annotations related to cancer, amongst others, and both have functional links to MYC regulation. Genomic signatures identified in lymphoma cases suggest that increased risk contributed by the regions identified by GWAS may complement a complex predisposing genetic background.