PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections.

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

Treatment of Relapsing HPV Diseases by Restored Function of Natural Killer Cells.

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (IL2RG), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).

PET imaging of Aspergillus infection using Zirconium-89 labeled anti-ß-glucan antibody fragments.

PURPOSE: Invasive fungal diseases, such as pulmonary aspergillosis, are common life-threatening infections in immunocompromised patients and effective treatment is often hampered by delays in timely and specific diagnosis. Fungal-specific molecular imaging ligands can provide non-invasive readouts of deep-seated fungal pathologies. In this study, the utility of antibodies and antibody fragments (Fab) targeting ß-glucans in the fungal cell wall to detect Aspergillus infections was evaluated both in vitro and in preclinical mouse models. METHODS: The binding characteristics of two commercially available ß-glucan antibody clones and their respective antigen-binding Fabs were tested using biolayer interferometry (BLI) assays and immunofluorescence staining. In vivo binding of the Zirconium-89 labeled antibodies/Fabs to fungal pathogens was then evaluated using PET/CT imaging in mouse models of fungal infection, bacterial infection and sterile inflammation. RESULTS: One of the evaluated antibodies (HA-ßG-Ab) and its Fab (HA-ßG-Fab) bound to ß-glucans with high affinity (KD = 0.056 & 21.5 nM respectively). Binding to the fungal cell wall was validated by immunofluorescence staining and in vitro binding assays. ImmunoPET imaging with intact antibodies however showed slow clearance and high background signal as well as nonspecific accumulation in sites of infection/inflammation. Conversely, specific binding of [89Zr]Zr-DFO-HA-ßG-Fab to sites of fungal infection was observed when compared to the isotype control Fab and was significantly higher in fungal infection than in bacterial infection or sterile inflammation. CONCLUSIONS: [89Zr]Zr-DFO-HA-ßG-Fab can be used to detect fungal infections in vivo. Targeting distinct components of the fungal cell wall is a viable approach to developing fungal-specific PET tracers.

Persistent GDNF Expression 45 Months after Putaminal Infusion of AAV2-GDNF in a Patient with Parkinson's Disease.

OBJECTIVE: Gene therapy by convection-enhanced delivery of type 2 adeno-associated virus-glial cell derived neurotrophic factor (AAV2-GDNF) to the bilateral putamina seeks to increase GDNF gene expression and treat Parkinson's disease (PD). METHODS: A 63-year-old man with advanced PD received AAV2-GDNF in a clinical trial. He died from pneumonia after anterior cervical discectomy and fusion 45 months later. An autopsy included brain examination for GDNF transgene expression. Putaminal catecholamine concentrations were compared to in vivo 18F-Fluorodopa (18F-FDOPA) positron emission tomography (PET) scanning results before and 18 months after AAV2-GDNF infusion. RESULTS: Parkinsonian progression stabilized clinically. Postmortem neuropathology confirmed PD. Bilateral putaminal regions previously infused with AAV2-GDNF expressed the GDNF gene. Total putaminal dopamine was 1% of control, confirming the striatal dopaminergic deficiency suggested by baseline 18F-DOPA-PET scanning. Putaminal regions responded as expected to AAV2-GDNF. CONCLUSION: After AAV2-GDNF infusion, infused putaminal regions showed increased GDNF gene expression, tyrosine hydroxylase immunoreactive sprouting, catechol levels, and 18F-FDOPA-PET signal, suggesting the regenerative potential of AAV2-GDNF in PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Molecular Imaging of Infections: Advancing the Search for the Hidden Enemy.

Even before the coronavirus disease 2019 pandemic, infections were a major threat to human health, as the third leading cause of death and the leading cause of morbidity among all human diseases. Although conventional imaging studies are routinely used for patients with infections, they provide structural or anatomic information only. Molecular imaging technologies enable noninvasive visualization of molecular processes at the cellular level within intact living subjects, including patients, and hold great potential for infections. We hope that this supplement will spur interest in the field and establish new collaborations to develop and translate novel molecular imaging approaches to the clinic.

Radiotracer Development for Fungal-Specific Imaging: Past, Present, and Future.

Invasive fungal infections have become a major challenge for public health, mainly due to the growing numbers of immunocompromised patients, with high morbidity and mortality. Currently, conventional imaging modalities such as computed tomography and magnetic resonance imaging contribute largely to the noninvasive diagnosis and treatment evaluation of those infections. These techniques, however, often fall short when a fast, noninvasive and specific diagnosis of fungal infection is necessary. Molecular imaging, especially using nuclear medicine-based techniques, aims to develop fungal-specific radiotracers that can be tested in preclinical models and eventually translated to human applications. In the last few decades, multiple radioligands have been developed and tested as potential fungal-specific tracers. These include radiolabeled peptides, antifungal drugs, siderophores, fungal-specific antibodies, and sugars. In this review, we provide an overview of the pros and cons of the available radiotracers. We also address the future prospects of fungal-specific imaging.

Advances and Challenges in Molecular Imaging of Viral Infections.

Molecular imaging of viral infection, using a variety of advanced imaging techniques such as optical and nuclear imaging, can and has been used for direct visualization of the virus as well as assessment of virus-host interactions. Unlike imaging of other pathogens such as bacteria and fungi, challenging aspects of imaging viral infections include the small size of viruses, the complexity of viral infection animal models (eg, species dependence), and the high-level containment needs for many high-consequence pathogens, among others. In this review, using representative viral infections, we discuss how molecular imaging can reveal real-time infection dynamics, improve our understanding of disease pathogenesis, and guide optimization of treatment and prevention strategies. Key findings from human and animal studies are highlighted.

The Promise of Molecular Imaging: Focus on Central Nervous System Infections.

Central nervous system (CNS) infections can lead to high mortality and severe morbidity. Diagnosis, monitoring, and assessing response to therapy of CNS infections is particularly challenging with traditional tools, such as microbiology, due to the dangers associated with invasive CNS procedures (ie, biopsy or surgical resection) to obtain tissues. Molecular imaging techniques like positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging have long been used to complement anatomic imaging such as computed tomography (CT) and magnetic resonance imaging (MRI), for in vivo evaluation of disease pathophysiology, progression, and treatment response. In this review, we detail the use of molecular imaging to delineate host-pathogen interactions, elucidate antimicrobial pharmacokinetics, and monitor treatment response. We also discuss the utility of pathogen-specific radiotracers to accurately diagnose CNS infections and strategies to develop radiotracers that would cross the blood-brain barrier.

Imaging of Brain Structural and Functional Effects in People With Human Immunodeficiency Virus.

Before the introduction of antiretroviral therapy, human immunodeficiency virus (HIV) infection was often accompanied by central nervous system (CNS) opportunistic infections and HIV encephalopathy marked by profound structural and functional alterations detectable with neuroimaging. Treatment with antiretroviral therapy nearly eliminated CNS opportunistic infections, while neuropsychiatric impairment and peripheral nerve and organ damage have persisted among virally suppressed people with HIV (PWH), suggesting ongoing brain injury. Neuroimaging research must use methods sensitive for detecting subtle HIV-associated brain structural and functional abnormalities, while allowing for adjustments for potential confounders, such as age, sex, substance use, hepatitis C coinfection, cardiovascular risk, and others. Here, we review existing and emerging neuroimaging tools that demonstrated promise in detecting markers of HIV-associated brain pathology and explore strategies to study the impact of potential confounding factors on these brain measures. We emphasize neuroimaging approaches that may be used in parallel to gather complementary information, allowing efficient detection and interpretation of altered brain structure and function associated with suboptimal clinical outcomes among virally suppressed PWH. We examine the advantages of each imaging modality and systematic approaches in study design and analysis. We also consider advantages of combining experimental and statistical control techniques to improve sensitivity and specificity of biotype identification and explore the costs and benefits of aggregating data from multiple studies to achieve larger sample sizes, enabling use of emerging methods for combining and analyzing large, multifaceted data sets. Many of the topics addressed in this article were discussed at the National Institute of Mental Health meeting "Biotypes of CNS Complications in People Living with HIV," held in October 2021, and are part of ongoing research initiatives to define the role of neuroimaging in emerging alternative approaches to identifying biotypes of CNS complications in PWH. An outcome of these considerations may be the development of a common neuroimaging protocol available for researchers to use in future studies examining neurological changes in the brains of PWH.

Tocilizumab as a Potential Adjunctive Therapy to Corticosteroids in Cryptococcal Post-infectious Inflammatory Response Syndrome (PIIRS): a Report of Two Cases.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Tocilizumab may be a safe adjunctive treatment for CM-related PIIRS suggesting further study.

Persistent neurological symptoms and elevated intracranial pressures in a previously healthy host with cryptococcal meningitis.

Cryptococcal meningoencephalitis can occur in both previously healthy and immunocompromised hosts. Here, we describe a 55 year-old HIV-negative male with no known prior medical problems, who presented with three months of worsening headaches, confusion, and memory changes without fever. Magnetic resonance imaging of the brain demonstrated bilateral enlargement/enhancement of the choroid plexi, with hydrocephalus, temporal and occipital horn entrapments, as well as marked periventricular transependymal cerebrospinal fluid (CSF) seepage. CSF analysis yielded a lymphocytic pleocytosis and cryptococcal antigen titer of 1:160 but sterile fungal cultures. Despite standard antifungal therapy and CSF drainage, the patient had worsening confusion and persistently elevated intracranial pressures. External ventricular drainage led to improved mental status but only with valve settings at negative values. Ventriculoperitoneal shunt placement could thus not be considered due to a requirement for drainage into the positive pressure venous system. Due to this persistent CSF inflammation and cerebral circulation obstruction, the patient required transfer to the National Institute of Health. He was treated for cryptococcal post-infectious inflammatory response syndrome with pulse-taper corticosteroid therapy, with resultant reductions in CSF pressures along with decreased protein and obstructive material, allowing successful shunt placement. After tapering of corticosteroids, the patient recovered without sequelae. This case highlights (1) the necessity to consider cryptococcal meningitis as a rare cause of neurological deterioration in the absence of fever even in apparently immunocompetent individuals and (2) the potential for obstructive phenomena from inflammatory sequelae and the prompt response to corticosteroid therapy.

Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

To Induce Immune Reconstitution Inflammatory Syndrome or Suppress It: The Spectrum of Mycobacterium genavense in the Antiretroviral Era.

Mycobacterium  genavense is a challenging opportunistic pathogen to diagnose and manage in patients with human immunodeficiency virus (HIV). Persistent immunosuppression or protracted immune reconstitution inflammatory syndrome can lead to complicated clinical courses. We describe 3 cases of M. genavense in patients with HIV representing the spectrum between disease burden and strength of immune response.

Outcomes in Previously Healthy Cryptococcal Meningoencephalitis Patients Treated With Pulse Taper Corticosteroids for Post-infectious Inflammatory Syndrome.

BACKGROUND: Cryptococcal meningoencephalitis (CM) is a major cause of mortality in immunosuppressed patients and previously healthy individuals. In the latter, a post-infectious inflammatory response syndrome (PIIRS) is associated with poor clinical response despite antifungal therapy and negative cerebrospinal fluid (CSF) cultures. Data on effective treatment are limited. METHODS: Between March 2015 and March 2020, 15 consecutive previously healthy patients with CM and PIIRS were treated with adjunctive pulse corticosteroid taper therapy (PCT) consisting of intravenous methylprednisolone 1 gm daily for 1 week followed by oral prednisone 1 mg/kg/day, tapered based on clinical and radiological response plus oral fluconazole. Montreal cognitive assessments (MOCA), Karnofsky performance scores, magnetic resonance imaging (MRI) brain scanning, ophthalmic and audiologic exams, and CSF parameters including cellular and soluble immune responses were compared at PIIRS diagnosis and after methylprednisolone completion. RESULTS: The median time from antifungal treatment to steroid initiation was 6 weeks. The most common symptoms at PIIRS diagnosis were altered mental status and vision changes. All patients demonstrated significant improvements in MOCA and Karnofsky scores at 1 month (P < .0003), which was accompanied by improvements in CSF glucose, white blood cell (WBC) count, protein, cellular and soluble inflammatory markers 1 week after receiving corticosteroids (CS) (P < .003). All patients with papilledema and visual field deficits also exhibited improvement (P < .0005). Five out of 7 patients who underwent audiological testing demonstrated hearing improvement. Brain MRI showed significant improvement of radiological findings (P = .001). CSF cultures remained negative. CONCLUSIONS: PCT in this small cohort of PIIRS was associated with improvements in CM-related complications with minimal toxicity in the acute setting.

An Unusual Pattern of Premature Cervical Spine Degeneration in STAT3-LOF.

Loss of function mutations in STAT3 (STAT3-LOF; autosomal dominant hyper-IgE (Job's) syndrome) are associated with a variety of musculoskeletal manifestations, including scoliosis, osteoporosis, and minimal trauma fractures. This retrospective magnetic resonance (MR) imaging study sought to characterize an unusual pattern of cervical spine degeneration among a cohort of STAT3-LOF patients. Cervical spine MR images of the STAT3-LOF cohort (n = 38) were assessed for a variety of degenerative changes and compared to age-matched groups of controls (n = 42) without known immune or musculoskeletal abnormalities. A unique pattern of premature cervical spine degeneration was identified among the STAT3-LOF cohort which included straightening and eventual reversal of the normal cervical lordosis, mainly due to multi-level spondylolisthesis, as well as early development of spinal canal narrowing, cord compression, and myelomalacia. Cervical spine degeneration in the STAT3-LOF cohort was significantly worse than controls in both the 30-45 and 45 + age groups. Moderate to severe degenerative changes were present after age 30, and markedly worsened over time in several cases. Bone mineral density (BMD) had a moderate negative correlation with cervical degeneration severity and a strong negative correlation with age among STAT3-LOF participants. Cervical degeneration in STAT3-LOF appears to be progressive and could result in cord compromise if left unaddressed. Focused history and physical examination for signs of neurologic compromise as well as periodic MR imaging are thus recommended for the evaluation of premature cervical spine degeneration in STAT3-LOF patients after age 30 so that timely surgical interventions may be considered to prevent spinal cord damage and permanent neurological deficits.

Central Nervous System Histoplasma-Associated Post-infectious Inflammatory Response Syndrome (Histo-PIIRS).

We present a case of central nervous system (CNS) histoplasmosis in a previously healthy adult with hepatitis C (HCV) presenting with neurological symptoms refractory to antifungal therapy and ventriculoperitoneal (VP) shunting 4 months after initial diagnosis. Persistent symptoms were thought to be inflammatory rather than infectious given negative cerebrospinal fluid (CSF) and serum fungal antigens. The patient promptly improved after initiation of corticosteroid therapy. Elevated CSF cytokines and regional enhancement on brain MRI resolved with corticosteroid treatment. This is the first case of Histoplasma-associated post-infectious inflammatory response syndrome (Histo-PIIRS) documented by CSF cytokine reduction in response to corticosteroid therapy.

Venetoclax/decitabine for a pediatric patient with chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome (MDS)/myeloproliferative disorder most commonly seen in the elderly. We describe an adolescent with monosomy 7 CMML presenting as central diabetes insipidus (DI), who was treated with venetoclax and decitabine as a bridge to hematopoietic stem cell transplantation (HSCT). Central DI is a rare manifestation of monosomy 7-associated MDS including CMML, itself a rare manifestation of GATA2 deficiency, particularly in children. Venetoclax/decitabine was effective for treatment of CMML as a bridge to HSCT.