Chikungunya Virus Vaccine Candidate Incorporating Synergistic Mutations Is Attenuated and Protects Against Virulent Virus Challenge.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus that periodically emerges to cause large epidemics of arthritic disease. Although the robust immunity elicited by live-attenuated virus (LAV) vaccine candidates makes them attractive, CHIKV vaccine development has been hampered by a high threshold for acceptable adverse events. METHODS: We evaluated the vaccine potential of a recently described LAV, skeletal muscle-restricted virus (SKE), that exhibits diminished replication in skeletal muscle due to insertion of target sequences for skeletal muscle-specific miR-206. We also evaluated whether these target sequences could augment safety of an LAV encoding a known attenuating mutation, E2 G82R. Attenuation of viruses containing these mutations was compared with a double mutant, SKE G82R. RESULTS: SKE was attenuated in both immunodeficient and immunocompetent mice and induced a robust neutralizing antibody response, indicating its vaccine potential. However, only SKE G82R elicited diminished swelling in immunocompetent mice at early time points postinoculation, indicating that these mutations synergistically enhance safety of the vaccine candidate. CONCLUSIONS: These data suggest that restriction of LAV replication in skeletal muscle enhances tolerability of reactogenic vaccine candidates and may improve the rational design of CHIKV vaccines.

Emerging Chikungunya Virus Variants at the E1-E1 Interglycoprotein Spike Interface Impact Virus Attachment and Inflammation.

Chikungunya virus (CHIKV) is a reemerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intrahost evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, cooccurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by antiglycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host. IMPORTANCE Alphaviruses represent a growing threat to human health worldwide. The reemerging alphavirus chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved vaccines or therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we identify and characterize the emerging E1 glycoprotein variants, V156A and K211T, that have since emerged in nature. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has only been attributed to specific residues of the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T increase foot-swelling of the ipsilateral foot in mice infected with these variants. Observing that these variants and other pathogenic variants occur at the E1-E1 interspike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further define the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.

EDP-938, a novel nucleoprotein inhibitor of respiratory syncytial virus, demonstrates potent antiviral activities in vitro and in a non-human primate model.

EDP-938 is a novel non-fusion replication inhibitor of respiratory syncytial virus (RSV). It is highly active against all RSV-A and B laboratory strains and clinical isolates tested in vitro in various cell lines and assays, with half-maximal effective concentrations (EC50s) of 21, 23 and 64 nM against Long (A), M37 (A) and VR-955 (B) strains, respectively, in the primary human bronchial epithelial cells (HBECs). EDP-938 inhibits RSV at a post-entry replication step of the viral life cycle as confirmed by time-of-addition study, and the activity appears to be mediated by viral nucleoprotein (N). In vitro resistance studies suggest that EDP-938 presents a higher barrier to resistance compared to viral fusion or non-nucleoside L polymerase inhibitors with no cross-resistance observed. Combinations of EDP-938 with other classes of RSV inhibitors lead to synergistic antiviral activity in vitro. Finally, EDP-938 has also been shown to be efficacious in vivo in a non-human primate model of RSV infection.

Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection.

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239.

EDP-239, a novel hepatitis C virus (HCV) inhibitor targeting nonstructural protein 5A (NS5A), has been investigated in vitro and in vivo EDP-239 is a potent, selective inhibitor with potency at picomolar to nanomolar concentrations against HCV genotypes 1 through 6. In the presence of human serum, the potency of EDP-239 was reduced by less than 4-fold. EDP-239 is additive to synergistic with other direct-acting antivirals (DAAs) or host-targeted antivirals (HTAs) in blocking HCV replication and suppresses the selection of resistance in vitro Furthermore, EDP-239 retains potency against known DAA- or HTA-resistant variants, with half-maximal effective concentrations (EC50s) equivalent to those for the wild type. In a phase I, single-ascending-dose, placebo-controlled clinical trial, EDP-239 demonstrated excellent pharmacokinetic properties that supported once daily dosing. A single 100-mg dose of EDP-239 resulted in reductions in HCV genotype 1a viral RNA of >3 log10 IU/ml within the first 48 h after dosing and reductions in genotype 1b viral RNA of >4-log10 IU/ml within 96 h. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

Tubarial gland sparing with intensity modulated radiation therapy for oropharyngeal cancers: A pilot study of dosimetric feasibility.

BACKGROUND: Tubarial glands are a new organ at risk for head and neck cancer radiation therapy (RT). We aimed to study the feasibility of sparing them using intensity-modulated radiation therapy (IMRT). METHODS: Tubarial glands were delineated for 17 patients with oropharyngeal carcinoma receiving definitive RT, and treatment plans were re-optimized to spare dose to the tubarial glands while maintaining target coverage. A paired t test was performed to compare the mean dose of tubarial glands and target coverage. RESULTS: The difference in mean doses was 4.9 and 7.0 Gy for the ipsilateral and contralateral tubarial glands, respectively (p < 0.01). The mean dose to tubarial gland was ≤39 Gy in 35% versus 47% (ipsilateral) and 70% versus 100% (contralateral) in clinical and re-optimized plans, respectively. Re-optimized ipsilateral tubarial gland mean ≤39 Gy was achieved more commonly in patients with base of tongue versus tonsil primaries (86% vs. 20%, p = 0.02). CONCLUSION: This pilot study demonstrates the dosimetric feasibility of tubarial gland sparing with IMRT. Dosimetric constraints need to be determined with larger studies.

Accelerated Hypofractionated Magnetic Resonance Guided Adaptive Radiation Therapy for Ultracentral Lung Tumors.

Radiotherapy for ultracentral lung tumors represents a treatment challenge, considering the high rates of high-grade treatment-related toxicities with stereotactic body radiation therapy (SBRT) or hypofractionated schedules. Accelerated hypofractionated magnetic resonance-guided adaptive radiation therapy (MRgART) emerged as a potential game-changer for tumors in these challenging locations, in close proximity to central organs at risk, such as the trachea, proximal bronchial tree, and esophagus. In this series, 13 consecutive patients, predominantly male (n = 9), with a median age of 71 (range (R): 46-85), underwent 195 MRgART fractions (all 60 Gy in 15 fractions) to metastatic (n = 12) or primary ultra-central lung tumors (n = 1). The median gross tumor volumes (GTVs) and planning target volumes (PTVs) were 20.72 cc (R: 0.54-121.65 cc) and 61.53 cc (R: 3.87-211.81 cc), respectively. The median beam-on time per fraction was 14 min. Adapted treatment plans were generated for all fractions, and indications included GTV/PTV undercoverage, OARs exceeding tolerance doses, or both indications in 46%, 18%, and 36% of fractions, respectively. Eight patients received concurrent systemic therapies, including immunotherapy (four), chemotherapy (two), and targeted therapy (two). The crude in-field loco-regional control rate was 92.3%. No CTCAE grade 3+ toxicities were observed. Our results offer promising insights, suggesting that MRgART has the potential to mitigate toxicities, enhance treatment precision, and improve overall patient care in the context of ultracentral lung tumors.

Clinical application of an institutional fractionated stereotactic radiosurgery (FSRS) program for brain metastases delivered with MRIdianⓇ BrainTx™.

Single-fraction stereotactic radiosurgery (SRS) or fractionated SRS (FSRS) are well established strategies for patients with limited brain metastases. A broad spectrum of modern dedicated platforms are currently available for delivering intracranial SRS/FSRS; however, SRS/FSRS delivered using traditional CT-based platforms relies on the need for diagnostic MR images to be coregistered to planning CT scans for target volume delineation. Additionally, the on-board image guidance on traditional platforms yields limited inter-fraction and intra-fraction real-time visualization of the tumor at the time of treatment delivery. MR Linacs are capable of obtaining treatment planning MR and on-table MR sequences to enable visualization of the targets and organs-at-risk and may subsequently help identify anatomical changes prior to treatment that may invoke the need for on table treatment adaptation. Recently, an MR-guided intracranial package (MRIdian A3i BrainTxTM) was released for intracranial treatment with the ability to perform high-resolution MR sequences using a dedicated brain coil and cranial immobilization system. The objective of this report is to provide, through the experience of our first patient treated, a comprehensive overview of the clinical application of our institutional program for FSRS adaptive delivery using MRIdian's A3i BrainTx system-highlights include reviewing the imaging sequence selection, workflow demonstration, and details in its delivery feasibility in clinical practice, and dosimetric outcomes.

Treatment of glioblastoma using MRIdian® A3i BrainTx™: Imaging and treatment workflow demonstration.

For patients with newly diagnosed glioblastoma, the current standard-of-care includes maximal safe resection, followed by concurrent chemoradiotherapy and adjuvant temozolomide, with tumor treating fields. Traditionally, diagnostic imaging is performed pre- and post-resection, without additional dedicated longitudinal imaging to evaluate tumor volumes or other treatment-related changes. However, the recent introduction of MR-guided radiotherapy using the ViewRay MRIdian A3i system includes a dedicated BrainTx package to facilitate the treatment of intracranial tumors and provides daily MR images. We present the first reported case of a glioblastoma imaged and treated using this workflow. In this case, a 67-year-old woman underwent adjuvant chemoradiotherapy after gross total resection of a left frontal glioblastoma. The radiotherapy treatment plan consisted of a traditional two-phase design (46 Gy followed by a sequential boost to a total dose of 60 Gy at 2 Gy/fraction). The treatment planning process, institutional workflow, treatment imaging, treatment timelines, and target volume changes visualized during treatment are presented. This case example using our institutional A3i system workflow successfully allows for imaging and treatment of primary brain tumors and has the potential for margin reduction, detection of early disease progression, or to detect the need for dose adaptation due to interfraction tumor volume changes.

Clinical applications of digital 2-D and 3-D radiography for the periodontist.

CONTEXT: Although there has been rapid development of imaging technology within dentistry, there has been limited evidence-based research documenting the benefits of digital radiography. EVIDENCE: We searched MEDLINE for relevant studies and review papers demonstrating clinical applications, limitations, and advancements within digital radiography. EVIDENCE SYNTHESIS: Two-dimensional (2-D) and 3-D digital radiography has become a powerful diagnostic tool for simple and complex procedures, including implant reconstruction. Recent advancements have reduced radiation exposure, increased resolution, and improved detection capabilities of complementary metal oxide semiconductor (CMOS) and cone-beam computed tomography (CBCT) sensors. The current review summarizes such advances and outlines advanced CBCT implant-planning techniques. CONCLUSIONS: While evidence-based research grows, the logistic, diagnostic, and planning improvements of 2-D and 3-D digital radiography are irrefutable with the potential to supplant conventional techniques.

Pulsed-Reduced Dose Rate (PRDR) Radiotherapy for Recurrent Primary Central Nervous System Malignancies: Dosimetric and Clinical Results.

PURPOSE: The objective was to describe PRDR outcomes and report EQD2 OAR toxicity thresholds. METHODS: Eighteen patients with recurrent primary CNS tumors treated with PRDR at a single institution between April 2017 and September 2021 were evaluated. The radiotherapy details, cumulative OAR doses, progression-free survival (PFS), overall survival (OS), and toxicities were collected. RESULTS: The median PRDR dose was 45 Gy (range: 36-59.4 Gy); the median cumulative EQD2 prescription dose was 102.7 Gy (range: 93.8-120.4 Gy). The median cumulative EQD2 D0.03cc for the brain was 111.4 Gy (range: 82.4-175.2 Gy). Symptomatic radiation necrosis occurred in three patients, for which the median EQD2 brain D0.03cc was 115.9 Gy (110.4-156.7 Gy). The median PFS and OS after PRDR were 6.3 months (95%CI: 0.9-11.6 months) and 8.6 months (95%CI: 4.9-12.3 months), respectively. The systematic review identified five peer-reviewed studies with a median cumulative EQD2 prescription dose of 110.3 Gy. At a median follow-up of 8.7 months, the median PFS and OS were 5.7 months (95%CI: 2.1-15.4 months) and 6.7 months (95%CI: 3.2-14.2 months), respectively. CONCLUSION: PRDR re-irradiation is a relatively safe and feasible treatment for recurrent primary CNS tumors. Despite high cumulative dose to OARs, the risk of high-grade, treatment-related toxicity within the first year of follow-up remains acceptable.

Submandibular gland transfer for the prevention of radiation-induced xerostomia in oropharyngeal cancer: Dosimetric impact in the intensity modulated radiotherapy era.

BACKGROUND: Submandibular gland (SMG) transfer decreased radiation-associated xerostomia in the 2/3-dimensional radiotherapy era. We evaluated the dosimetric implications of SMG transfer on modern intensity modulated radiotherapy (IMRT) plans. METHODS: Eighteen oropharynx cancer patients underwent SMG transfer followed by IMRT; reoptimized plans using the baseline SMG location were generated. Mean salivary gland, oral cavity, and larynx doses were compared between clinical plans and reoptimized plans. RESULTS: No statistically significant difference in mean SMG dose (27.53 Gy vs. 29.61 Gy) or total salivary gland dose (26.12 Gy vs. 26.41 Gy) was observed with or without SMG transfer (all p > 0.05). Mean oral cavity and larynx doses were not statistically different. Neither tumor site, target volume crossing midline, stage, nor salivary gland volumes were associated with mean doses. CONCLUSIONS: Salivary gland doses were similar with or without SMG transfer. IMRT likely decreases the benefit of SMG transfer on the risk of radiation-associated xerostomia.

Initial clinical experience with magnetic resonance-guided radiotherapy in pediatric patients: Lessons learned from a single institution with proton therapy.

Purpose/Objectives: Magnetic resonance-guided radiotherapy (MRgRT) is increasingly used in a variety of adult cancers. To date, published experience regarding the use of MRgRT in pediatric patients is limited to two case reports. We report on the use of MRgRT for pediatric patients at our institution during a four-year period and describe important considerations in the selection and application of this technology in children. Materials/Methods: All patients treated with MRgRT since inception at our institution between 4/2018 and 4/2022 were retrospectively reviewed. We also evaluated all pediatric patients treated at our institution during the same period who received either imaging or treatment using our magnetic resonance-guided linear accelerator (MR Linac). We summarize four clinical cases where MRgRT was selected for treatment in our clinic, including disease outcomes and toxicities and describe our experience using the MR Linac for imaging before and during treatment for image fusion and tumor assessments. Results: Between 4/2018 and 4/2022, 535 patients received MRgRT at our center, including 405 (75.7%) with stereotactic ablative radiotherapy (SABR). During this period, 347 distinct radiotherapy courses were delivered to pediatric patients, including 217 (62.5%) with proton therapy. Four pediatric patients received MRgRT. One received SABR for lung metastasis with daily adaptive replanning and a second was treated for liver metastasis using a non-adaptive workflow. Two patients received fractionated MRgRT for an ALK-rearranged non-small cell lung cancer and neuroblastoma. No Grade 2 or higher toxicities were observed or reported during MRgRT or subsequent follow-up. Twelve patients underwent MR imaging without contrast during treatment for brain tumors to assess for tumor/cystic changes. Two patients treated with other modalities underwent MR simulation for target volume delineation and organ at risk sparing due to anatomic changes during treatment or unexpected delays in obtaining diagnostic MR appointments. Conclusions: In four pediatric patients treated with MRgRT, treatment was well tolerated with no severe acute effects. At our center, most pediatric patients are treated with proton therapy, but the cases selected for MRgRT demonstrated significant organ at risk sparing compared to alternative modalities. In particular, MRgRT may provide advantages for thoracic/abdominal/pelvic targets using gated delivery and adaptive replanning, but selected patients treated with fractionated radiotherapy may also benefit MRgRT through superior organ at risk sparing.

Chikungunya virus replication in skeletal muscle cells is required for disease development.

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell-specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of Il1b, Il6, Ip10, and Tnfa transcripts. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

A soil bacterium alters sex determination and rhizoid development in gametophytes of the fern Ceratopteris richardii.

Gametophytes of the fern Ceratopteris richardii develop into either hermaphrodites or males. As hermaphrodites develop, they secrete antheridiogen, or ACE, into the environment, inducing male development in undifferentiated gametophytes. Hermaphrodites are composed of archegonia, antheridia, rhizoids and a notch meristem, while males consist of antheridia and rhizoids. Much of the research on sexual and morphological development concerns gametophytes grown in sterile environments. Using biochemical and molecular techniques we identify a soil bacterium and explore its effects on sexual and rhizoid development. Hermaphrodite and male gametophytes were exposed to this bacterium and the effects on sexual development, rhizoid length and rhizoid number were explored. The bacterium was identified as a pseudomonad, Pseudomonas nitroreducens. Gametophytes grown in the presence of the pseudomonad were more likely to develop into hermaphrodites across all gametophyte densities. Across all gametophyte sizes, hermaphrodites had rhizoids that were 2.95× longer in the presence of the pseudomonad while males had rhizoids that were 2.72× longer in the presence of the pseudomonad. Both hermaphrodite and male gametophytes developed fewer rhizoids in the presence of the pseudomonad. Control hermaphrodites produced 1.23× more rhizoids across all gametophyte sizes. For male gametophytes grown in the absence of the pseudomonad, the rate of increase in the number of rhizoids was greater with increasing size in the control than the rate of increase in males grown in the presence of the pseudomonad. The pseudomonad may be acting on gametophyte sexual development via several potential mechanisms: degradation of ACE, changes in nutrient availability or phytohormone production. The pseudomonad may also increase rhizoid number through production of phytohormones or changes in nutrient availability.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.