Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer and in this process some "old" biomarkers are re-utilized and given a new purpose. It is not always recognized that, by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory developed tests (LDTs), of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance (CBQA) Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory (USA) was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays (TMAs) with 32 cases and performed their in-house Ki-67 assay. The results were assessed using QuPath, an open-source software for bio-image analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20% and 30% cut-offs. Overall, PPA and NPA varied depending on the selected cut-off; participants were more successful with 5% and 10%, than with 20% and 30% cut-offs. Only four out of 16 laboratories had robust IHC protocols with acceptable PPA for all cut-offs. The lowest PPA for the 5% cut-off was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cut-off was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cut-offs. The poor agreement was not due to the readout, but rather due to IHC protocol conditions. IKWG recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Introduction: Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Methods: Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Results: Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Conclusion: Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson's disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.

Immunohistochemistry and immunofluorescence utilization audit by subspecialty in an academic setting: A step toward stewardship.

There are immunohistochemistry (IHC) and immunofluorescence (IF) panels described in the literature and established by personal and institutional experiences that are in common use by pathologists in their daily practice. Stewardship is a difficult discussion because IHC utilization is influenced by many factors including the pathologist's experience, background, practice setting, personal bias, and medicolegal culture. We developed the methodology to audit the IHC/IF utilization in our academic subspecialty practice. We aim to share this methodology and to provide our data that can be used for consideration by other subspecialized academic practices. This analysis included a total of 63,157 specimens that were accessioned during 2022, representing 38,612 cases. The likelihood of ordering IHC/IF ranged from 1 % (in genitourinary pathology) to 59 % (in renal pathology). The average percentage of specimens with IHC/IF was 21 % for the entire practice. In cases where IHC/IF was ordered, the number of stained slides averaged 4.9 per specimen for the entire practice. The number of IHC/IF slides per specimen ranged from 1.9 (in gastrointestinal pathology) to 12.2 (in renal pathology). The highest number of antibodies ordered for a single specimen by subspecialty ranged from 11 (in cardiac pathology) to 63 (in dermatopathology). Renal pathology was the only subspecialty that had an average number of IHC/IF slides that was statistically significantly different from all other subspecialties. We described the various patterns of utilization by subspecialty and rationalized their subtle differences. We also analyzed the types of cases that exceeded the reimbursement limits set by the Centers for Medicare and Medicaid Services (CMS).

27-Hydroxycholesterol Drives the Spread of α-Synuclein Pathology in Parkinson's Disease.

BACKGROUND: The accumulation and aggregation of α-synuclein (α-Syn) are characteristic of Parkinson's disease (PD). Epidemiological evidence indicates that hyperlipidemia is associated with an increased risk of PD. The levels of 27-hydroxycholesterol (27-OHC), a cholesterol oxidation derivative, are increased in the brain and cerebrospinal fluid of patients with PD. However, whether 27-OHC plays a role in α-Syn aggregation and propagation remains elusive. OBJECTIVE: The aim of this study was to determine whether 27-OHC regulates α-Syn aggregation and propagation. METHODS: Purified recombinant α-Syn, neuronal cultures, and α-Syn fibril-injected mouse model of PD were treated with 27-OHC. In addition, CYP27A1 knockout mice were used to investigate the effect of lowering 27-OHC on α-Syn pathology in vivo. RESULTS: 27-OHC accelerates the aggregation of α-Syn and enhances the seeding activity of α-Syn fibrils. Furthermore, the 27-OHC-modified α-Syn fibrils localize to the mitochondria and induce mitochondrial dysfunction and neurotoxicity. Injection of 27-OHC-modified α-Syn fibrils induces enhanced spread of α-Syn pathology and dopaminergic neurodegeneration compared with pure α-Syn fibrils. Similarly, subcutaneous administration of 27-OHC facilitates the seeding of α-Syn pathology. Genetic deletion of cytochrome P450 27A1 (CYP27A1), the enzyme that converts cholesterol to 27-OHC, ameliorates the spread of pathologic α-Syn, degeneration of the nigrostriatal dopaminergic pathway, and motor impairments. These results indicate that the cholesterol metabolite 27-OHC plays an important role in the pathogenesis of PD. CONCLUSIONS: 27-OHC promotes the aggregation and spread of α-Syn. Strategies aimed at inhibiting the CYP27A1-27-OHC axis may hold promise as a disease-modifying therapy to halt the progression of α-Syn pathology in PD. © 2023 International Parkinson and Movement Disorder Society.

CXCR4 antagonists disrupt leukaemia-meningeal cell adhesion and attenuate chemoresistance.

The effective prophylaxis and treatment of central nervous system (CNS) involvement in acute lymphoblastic leukaemia (ALL) remains a significant clinical challenge. Developing novel and more effective CNS-directed therapies has been hampered, in part, by our limited understanding of the leukaemia niche in the CNS relative to the bone marrow. Accordingly, defining the molecular and cellular components critical for the establishment and maintenance of the CNS leukaemia niche may lead to new therapeutic opportunities. In prior work we showed that direct intercellular interactions between leukaemia and meningeal cells enhance leukaemia chemoresistance in the CNS. Herein, we show that the CXCR4/CXCL12 chemokine axis contributes to leukaemia-meningeal cell adhesion. Importantly, clinically tested CXCR4 antagonists, which are likely to cross the blood-brain and blood-cerebral spinal fluid barriers and penetrate the CNS, effectively disrupted leukaemia-meningeal cell adhesion. Moreover, by disrupting these intercellular interactions, CXCR4 antagonists attenuated leukaemia chemoresistance in leukaemia-meningeal cell co-culture experiments and enhanced the efficacy of cytarabine in targeting leukaemia cells in the meninges in vivo. This work identifies the CXCR4/CXCL12 axis as an important regulator of intercellular interactions within the CNS leukaemia niche and supports further testing of the therapeutic efficacy of CXCR4 antagonists in overcoming CNS niche-mediated chemoresistance.

Galectin-9/Tim-3 pathway mediates dopaminergic neurodegeneration in MPTP-induced mouse model of Parkinson's disease.

Galectin-9 (Gal-9) is a crucial immunoregulatory mediator in the central nervous system. Microglial activation and neuroinflammation play a key role in the degeneration of dopaminergic neurons in the substantia nigra (SN) in Parkinson's disease (PD). However, it remains unknown whether Gal-9 is involved in the pathogenesis of PD. We found that MPP+ treatment promoted the expression of Gal-9 and pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α, and MIP-1α) in a concentration-dependent manner in BV2 cells. Gal-9 enhanced neurodegeneration and oxidative stress induced by MPP+ in SH-SY5Y cells and primary neurons. Importantly, deletion of Gal-9 or blockade of Tim-3 ameliorated microglial activation, reduced dopaminergic neuronal loss, and improved motor performance in an MPTP-induced mouse model of PD. These observations demonstrate a pathogenic role of the Gal-9/Tim-3 pathway in exacerbating microglial activation, neuroinflammation, oxidative stress, and dopaminergic neurodegeneration in the pathogenesis of PD.

Ubiquitin-positive astrogliopathy clinically mimicking Parkinson's disease.

Several neurodegenerative pathologies can clinically mimic Parkinson's disease, including neurodegenerative diseases with glial pathology. However, the glial aggregates are typically composed of known pathogenic proteins and are associated with prominent neuronal loss in the substantia nigra. Here we present an unusual case of a 91-year-old man with a clinical diagnosis of Parkinson's disease, but whose autopsy findings showed a ubiquitin-positive astrogliopathy without significant neuronal loss in the substantia nigra. These glial aggregates affected the basal ganglia, cortex, and cerebellum, and were negative for tau, alpha-synuclein, TDP-43, FUS, and p62. This case is a rare example of an unknown glial neurodegenerative pathology mimicking Parkinson's disease without significant loss of nigral dopaminergic neurons.

Initial Clinical Experience With ClearPoint SmartFrame Array-Aided Stereotactic Procedures.

BACKGROUND: The ClearPoint SmartFrame Array (ClearPoint Neuro, Inc., Solana Beach, CA) system consists of a magnetic resonance imaging compatible frame supported by a customized neuro navigation software. This system received U.S. Food and Drug Administrationclearance for clinical use in January 2021. Our objective was to report initial safety data and user experience of SmartFrame Array-supported stereotactic procedures. METHODS: We prospectively followed the first 10 consecutive patients who underwent stereotactic procedures supported by SmartFrame Array. Clinical and procedural data were recorded and compared to data obtained from prior cases with SmartFrame XG. RESULTS: Ten patients underwent stereotactic needle biopsy, stereotactic laser ablation (SLA), or combined biopsy/SLA procedures. For needle biopsies (n = 9), the average maximal diameter of the contrast-enhancing target lesion was 9.9 ± 2.8 mm. The radial error of stereotaxis was less than 2 mm. Definitive diagnosis was achieved in all cases. For procedures involving SLA (n = 5), 100% of the contrast-enhancing lesion was ablated. All patients were discharged home by postoperative day 2. There were no 30-day readmissions, morbidity, or mortality. The average stereotaxis time for the SmartFrame Array-aided single trajectory procedure was 80 ± 9.5 minutes, which compared favorably to that required for the earlier generation SmartFrame XG frame (111.5 ± 16.5 minutes; P < 0.01). The unique Array design supported stereotactic procedures that cannot be easily achieved with the previous SmartFrame XG frame. CONCLUSIONS: The SmartFrame Array system offers a more rigid and compact build to enhance procedural efficiency while maintaining accuracy and safety. The design supports multi-trajectory stereotaxis, allowing novel clinical applications.

GammaTile® brachytherapy in the treatment of recurrent glioblastomas.

BACKGROUND: GammaTile® (GT) is a recent U.S. Food and Drug Administration (FDA) cleared brachytherapy platform. Here, we report clinical outcomes for recurrent glioblastoma patients after GT treatment following maximal safe resection. METHODS: We prospectively followed twenty-two consecutive Isocitrate Dehydrogenase (IDH) wild-type glioblastoma patients (6 O6-Methylguanine-DNA methyltransferase methylated (MGMTm); sixteen MGMT unmethylated (MGMTu)) who underwent maximal safe resection of recurrent tumor followed by GT placement. RESULTS: The cohort consisted of 14 second and eight third recurrences. In terms of procedural safety, there was one 30-day re-admission (4.5%) for an incisional cerebrospinal fluid leak, which resolved with lumbar drainage. No other wound complications were observed. Six patients (27.2%) declined in Karnofsky Performance Score (KPS) after surgery due to worsening existing deficits. One patient suffered a new-onset seizure postsurgery (4.5%). There was one (4.5%) 30-day mortality from intracranial hemorrhage secondary to heparinization for an ischemic limb. The mean follow-up was 733 days (range 279-1775) from the time of initial diagnosis. Six-month local control (LC6) and twelve-month local control (LC12) were 86 and 81%, respectively. Median progression-free survival (PFS) was comparable for MGMTu and MGMTm patients (~8.0 months). Median overall survival (OS) was 20.0 months for the MGMTu patients and 37.4 months for MGMTm patients. These outcomes compared favorably to data in the published literature and an independent glioblastoma cohort of comparable patients without GT treatment. CONCLUSIONS: This clinical experience supports GT brachytherapy as a treatment option in a multi-modality treatment strategy for recurrent glioblastomas.

Modeling the effect of cerebral capillary blood flow on neuronal firing.

Adequate cerebral blood flow has long been recognized as essential for the maintenance of the neuronal function while interruption of cerebral blood flow for durations as short as minutes can result in permanent brain damage. A primary goal of this work is to determine how a neuron's ability to respond to synaptic input depends on parameters that control cerebral blood flow. A complex mathematical model is constructed that integrates detailed biophysical models of neuronal action potentials, mitochondrial ATP production and cerebral capillary blood flow. The model also provides insights of the role of astrocytes in maintaining neuronal responses, as well as the impact of elevated cytosolic calcium, due to increased synaptic activity, on mitochondrial ATP production. Both dynamical systems analysis and numerical simulations are used to determine how the maximum frequency at which the neurons can respond to synaptic input depends on capillary blow flow, as well as the ability of astrocytes to buffer extracellular potassium and cytosolic calcium handling. Results are presented for both the cases of homogenous and heterogeneous capillary networks. These results demonstrate, through this interconnected model, that heterogeneity of the capillary flow results in a decrease in the ability of neurons to respond to synaptic stimulation and that intact glial function provides a further protective role for the neurons.

Molecular Profile of a Pituitary Rhabdomyosarcoma Arising From a Pituitary Macroadenoma: A Case Report.

Pituitary sarcoma arising in association with pituitary adenoma is an uncommon finding. Most cases of secondary sarcoma have been noted to arise with a median interval of 10.5 years post radiation. In this case report, we describe a 77-year-old man with an incidental discovery of a pituitary macroadenoma on magnetic resonance imaging (MRI) and underwent radiotherapy. Three years after radiation treatment, there was an acute change in clinical symptoms and increase in tumor size and mass effect on the optic chiasm which prompted surgical resection. A pituitary adenoma along with a separate spindle-cell sarcomatous component was identified in histology. Immunohistochemical stain for muscle markers confirmed a development of pituitary rhabdomyosarcoma (RMS). Molecular profiling of the tumor identified mutations in TP53, ATRX, LZTR1, and NF1. Despite its rarity, characterization of pituitary RMS with immunohistochemistry and molecular studies may provide an insight to its pathophysiological relationship with pituitary adenoma.

Mathematical modeling of cerebral capillary blood flow heterogeneity and its effect on brain tissue oxygen levels.

Maintaining cerebral blood flow is critical for adequate neuronal function. Previous computational models of brain capillary networks have predicted that heterogeneous cerebral capillary flow patterns result in lower brain tissue partial oxygen pressures PO2). However, these previous models have often considered simple capillary networks in terms of their geometric properties. In this current work, we developed and analyzed computational models of brain capillary networks to determine how perturbations of network properties impact tissue oxygen levels. The models include variabilities in both their geometric (segment lengths and diameters) and three-dimensional, topological structure. Two classes of capillary network models are considered. The first consists of equations for the oxygen partial pressure, PO2, in both a capillary network and the surrounding tissue. In order to gain insight into the behavior of this detailed model, we also consider a reduced model for changes in PO2 in just the capillary network. The main result is that for a general class of networks, random perturbations of either segment diameters or conductances will always, on average, decrease the average tissue oxygen levels. This result is supported through both simulations of the models and mathematical analysis. Our results promise to expand our understanding of cerebral capillary blood flow and its impact on the brain function in health and disease.

IDH1 and IDH2 Mutations in Colorectal Cancers.

OBJECTIVES: To elucidate clinicopathologic and molecular characteristics of IDH1 and IDH2 (IDH1/2) mutations in colorectal cancers (CRCs). METHODS: We evaluated IDH1/2 mutations in 1,623 CRCs using a next-generation sequencing assay. RESULTS: IDH1/2 mutations, predominantly IDH1 p.R132C, were detected in 15 (0.9%) CRCs and in 5 (3.0%) of 167 BRAF p.V600E-mutated CRCs. Three IDH1/2-mutated CRCs were associated with inflammatory bowel disease. They were significantly associated with old age, mucinous or signet ring cell adenocarcinoma, and high-grade histomorphology. Concordance of variant allele frequency between IDH1/2 mutants and other trunk drivers in CRCs and presence of IDH1/2 mutation in the adenoma and early adenocarcinoma indicated IDH1/2 mutations could be trunk drivers suitable for targeted therapy. CONCLUSIONS: IDH1/2 mutations in CRCs were uncommon but enriched in BRAF p.V600E-mutated CRCs and perhaps colitis-associated CRCs. Further studies on IDH1/2-mutated CRCs are needed to clarify their clinicopathologic features and implications for targeted therapy.

Cerebral Microbleeds and Intracranial Hemorrhages in Adult Patients on Extracorporeal Membrane Oxygenation-Autopsy Study.

Current studies lack robust information on the prevalence and associated factors of cerebral microbleeds in patients who underwent extracorporeal membrane oxygenation. DESIGN: Retrospective analysis. SETTING: We reviewed patients who underwent (extracorporeal membrane oxygenation) and subsequent brain autopsy with gross and microscopic examinations from January 2009 to December 2018 from a single tertiary center. PATIENTS: Twenty-five extracorporeal membrane oxygenation patients (median age, 53 yr; interquartile range, 36-61 yr; 17 women and 8 men) underwent brain autopsy. INTERVENTIONS: Descriptive analysis of neuropathologic findings. Cerebral microbleed was defined as a small focus (< 10 mm diameter) of accumulation of blood product in the brain tissue. Macrohemorrhage was defined as any of the grossly identifiable epidural, subdural, subarachnoid, or intraparenchymal hemorrhages larger than 10 mm. MEASUREMENT AND MAIN RESULTS: Of 25 (22 venoarterial extracorporeal membrane oxygenation; three venovenous extracorporeal membrane oxygenation), 15 patients (60%) were found to have cerebral microbleeds, whereas 13 (52%) had macrohemorrhages, of whom five (20%) had both. Overall, 92% of brains demonstrated the presence of either cerebral microbleeds or macrohemorrhages after extracorporeal membrane oxygenation support. Of the patients with cerebral microbleeds, lobar cerebral microbleeds (80%) occurred more frequently than deep cerebral microbleeds (60%), with 40% of patients having both types. The cases of macrohemorrhages consisted of one epidural (8%), two subdural (15%), and 10 subarachnoid hemorrhages (77%). In univariate analyses, the presence of macrohemorrhages was significantly associated with the presence of cerebral microbleeds (p = 0.03) with odds ratio of 0.13 (CI, 0.02-0.82). Age, sex, extracorporeal membrane oxygenation duration, extracorporeal membrane oxygenation type, use of aspirin or dialysis during extracorporeal membrane oxygenation support, bloodstream infections, hemoglobin, platelets, and coagulopathy profiles were not associated with cerebral microbleeds. CONCLUSIONS: In patients with postmortem neuropathologic evaluation, 92% sustained acute cerebral microbleeds or macrohemorrhages after extracorporeal membrane oxygenation support. Cerebral microbleeds were commonly present in the majority of extracorporeal membrane oxygenation nonsurvivors. Further research is necessary to study the long-term sequelae, such as cognitive outcome of extracorporeal membrane oxygenation-associated cerebral microbleeds in extracorporeal membrane oxygenation survivors.

Multiple Neurodegenerative Pathologies in an Alzheimer's Disease Patient Treated with Fornical Deep Brain Stimulation.

As an established treatment for movement disorders, deep brain stimulation (DBS) has been adapted for the treatment of Alzheimer's disease (AD) by modulating fornix activity. Although it is generally regarded as a safe intervention in patients over 65 years of age, the complex neurophysiology and interconnection within circuits connected to the fornix warrants a careful ongoing evaluation of the true benefit and risk potential of DBS on slowing cognitive decline in AD patients. Here we report on a patient who died long after being implanted with a DBS device who donated her brain for neuropathologic study. The autopsy confirmed multiple proteinopathies including AD-related change, diffuse neocortical Lewy body disease, TDP-43 proteinopathy, and a nonspecific tauopathy. We discuss the possible mechanisms of these overlapping neurodegenerative disorders and caution that future studies of DBS for AD will need to take these findings into consideration.

Associations of Alzheimer's Disease Neuropathologic Changes with Clinical Presentations of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) and Alzheimer's disease (AD) are the two most prevalent neurodegenerative diseases associated with age. Pathological studies have shown that these two diseases share a certain degree of neuropathological overlap. AD neuropathologic change contributes to cognitive impairment in PD. However, the impact of AD pathology on other clinical phenotypes in PD remains largely unknown. OBJECTIVE: Herein we aimed to assess the impact of co-occurring AD neuropathologic change on the clinical phenotypes of PD. METHODS: We examined 46 autopsy brains of PD patients and available clinical information to retrospectively assess the effects of comorbid AD pathology on dementia, hallucinations, and dyskinesia commonly seen in advanced PD. RESULTS: AD neuropathology significantly increased the risk of hallucinations and dementia, but not dyskinesia in PD patients. Surprisingly, diffuse Lewy body pathology, but not AD pathology, was associated with the occurrence of dementia and hallucinations. Most importantly, we reported that the severity of neuronal loss in the locus coeruleus (LC), but not the severity of neuronal loss in the substantia nigra (SN), was associated with the occurrence of dyskinesia in advanced PD patients, while neither Lewy body scores in SN nor LC had significant effects. CONCLUSION: We show for the first time that neuronal loss in LC contributes to dyskinesia. Understanding the relationships between the two distinct pathologies and their relevant clinical phenotypes will be crucial in the development of effective disease-modifying therapies for PD.

Necrotizing enterocolitis induces T lymphocyte-mediated injury in the developing mammalian brain.

Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4+ T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into Rag1 -/- recipient mice lacking CD4+ T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4+ T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4+ T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve Rag1 -/- recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4+ T lymphocytes into naïve Rag1 -/- recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ-releasing CD4+ T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.