Citations

Abstract Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our laboratory has developed a model of CD8 T cell-mediated BBB disruption which employs a variation of the Theiler’s murine encephalomyelitis virus (TMEV) infection. At seven days post TMEV infection, the majority of antiviral CD8 T cells recognize an immunodominant virus peptide, VP2121–130, in the context of the H-2Db class I molecule. Upon intravenous administration of this VP2 peptide at seven days post infection, Db:VP2121–130 epitope specific CD8 T cells induce BBB in a perforin dependent process. In this model, we addressed the role of microglia in tandem with CD8+ T cells. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, perforin-deficient mice failed to display BBB disruption and had significantly diminished microglial expression of MHC-II and CD68 in this model. This study demonstrates CD8 T cell promote microglia activation through a perforin-dependent process during BBB disruption. Importantly, microglia are activated concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.

Abstract We have previously shown (AAI2009) that dendritic cells (DC) contribute to the survival of long lived plasma cells in the bone marrow, which are important to maintain persistent antibody titres. What is not clear are the molecular interactions that are involved. Both normal and malignant plasma cells(like myeloma cells) express the T-cell co-stimulatory molecule CD28, and both interact with DCs in the bone marrow microenvironment. CD28 activation on MM triggers similar molecular partners downstream as observed in T-cells. Gene expression analysis of normal and malignant plasma cells shows that CD28 expression correlates with poor prognostic subgroups that are associated with specific genetic lesions. In our data, while activation of CD28 with antibodies or DC-CD80/CD86 (B7) protects myeloma cells against cell death, the ligation of B7 on DCs induces DC production of plasma cell survival factor IL-6, and immunosuppressive factor indoleamine 2,3-dioxygenase (IDO). Use of Lenalidomide (a common myeloma drug) to inhibit DC-B7 expression or anti-CD28(Fab)2 fragments to block CD28-B7 interactions reduced DC ability to protect MM cells. Experiments with αCD28(Fab)2 or inhibitors of notch pathway suggest a cross talk that regulates IL-6 production. IDO activity in DC-MM cocultures, suppressed T-cell proliferation in MLRs while an IDO inhibitor alleviated this. Our data suggests a role for CD28-B7 in plasma cell survival by modulating the bone marrow microenvironment.

Abstract Thymus organogenesis is dependent on insulin-like growth factor (IGF), but involuted thymi of old mice cannot be reconstituted with infusion of IGF. In adult humans, tumor growth is associated with local upregulation of IGF, but increased incidence of cancer with aging is associated with decreasing levels of serum IGF. We hypothesized that suppression of tissue availability of IGF prevents/delays thymic atrophy and maintains a pool of functionally competent T cells throughout life. Hence, we examined mice with homozygous deletion of PAPPA, a gene encoding for a protease that degrades inhibitory IGF-binding proteins (IGFBP) that limit IGF levels in tissues. PAPPA-/- mice have normal serum IGF levels, indicating maintenance of the endocrine IGF reservoir. They have 40% extension of lifespan; and have reduced burden of spontaneous tumors, the major cause of early mortality of wildtype mice. Strikingly, PAPPA-/- mice aged =18 months have histologically organized thymi densely populated by CD4+CD8+ thymocytes with high TREC levels. Bone marrows of old mutants are enriched with thymus-seeding progenitors. Old mutant mice have a diverse repertoire of T cells that are functionally indistinguishable from young wildtype mice. These data indicate an IGF-immune axis of longevity. We suggest that PAPPA-mediated proteolysis is a potential target for intervention to maintain thymic function and promote healthy aging.

Abstract Mouse alloantiserum capable of detecting minor histocompatibility antigens has been produced. Cytotoxic antibody to products of the H-4 locus was produced by reciprocal immunization of minor H loci congenic strains B10 (H-4a) and B10.129(21M) (H-4b), with mitogen-stimulated lymphocytes and epidermal cells. Absorption studies suggested antibodies to more than 1 specificity. The antibodies cross-react with products of other minor H loci. Alloantiserum to the H-4 gene product precipitates a glycoprotein with a m.w. of approximately 45,000 from radiolabeled Con A-stimulated lymph node cells.

Microglia play a critical role in the clearance of myelin debris, thereby ensuring functional recovery from neural injury. Here, using mouse model of demyelination following two-point LPC injection, we show that the microglial autophagic–lysosomal pathway becomes overactivated in response to severe demyelination, leading to lipid droplet accumulation and a dysfunctional and pro-inflammatory microglial state, and finally failed myelin debris clearance and spatial learning deficits. Data from genetic approaches and pharmacological modulations, via microglial Atg5 deficient mice and intraventricular BAF A1 administration, respectively, demonstrate that staged suppression of excessive autophagic–lysosomal activation in microglia, but not sustained inhibition, results in better myelin debris degradation and exerts protective effects against demyelination. Combined multi-omics results in vitro further showed that enhanced lipid metabolism, especially the activation of the linoleic acid pathway, underlies this protective effect. Supplementation with conjugated linoleic acid (CLA), both in vivo and in vitro, could mimic these effects, including attenuating inflammation and restoring microglial pro-regenerative properties, finally resulting in better recovery from demyelination injuries and improved spatial learning function, by activating the peroxisome proliferator-activated receptor (PPAR-γ) pathway. Therefore, we propose that pharmacological inhibition targeting microglial autophagic–lysosomal overactivation or supplementation with CLA could represent a potential therapeutic strategy in demyelinated disorders.

ObjectiveRecent evidence supports a link between increased TDP‐43 burden and the presence of an APOE4 gene allele in Alzheimer's disease (AD); however, it is difficult to conclude the direct effect of APOE on TDP‐43 pathology due to the presence of mixed AD pathologies. The goal of this study is to address how APOE isoforms impact TDP‐43 pathology and related neurodegeneration in the absence of typical AD pathologies.MethodsWe overexpressed human TDP‐43 via viral transduction in humanized APOE2, APOE3, APOE4 mice, and murine Apoe‐knockout (Apoe‐KO) mice. Behavior tests were performed across ages. Animals were harvested at 11 months of age and TDP‐43 overexpression‐related neurodegeneration and gliosis were assessed. To further address the human relevance, we analyzed the association of APOE with TDP‐43 pathology in 160 postmortem brains from autopsy‐confirmed amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD‐MND) in the Mayo Clinic Brain Bank.ResultsWe found that TDP‐43 overexpression induced motor function deficits, neuronal loss, and gliosis in the motor cortex, especially in APOE2 mice, with much milder or absent effects in APOE3, APOE4, or Apoe‐KO mice. In the motor cortex of the ALS and FTLD‐MND postmortem human brains, we found that the APOE2 allele was associated with more severe TDP‐43‐positive dystrophic neurites.InterpretationOur data suggest a genotype‐specific effect of APOE on TDP‐43 proteinopathy and neurodegeneration in the absence of AD pathology, with the strongest association seen with APOE2. ANN NEUROL 2023;93:830–843

AbstractBackgroundDigital radiography (DR) still presents many challenges and could have complex imaging acquisition and processing patterns in a clinical practice hindering quality standardization.PurposeThis technical note aims to report the 4‐year experience with utilizing a custom DICOM metadata analytics program in clinical DR at a large institution.MethodsThirty‐eight DR systems of three vendors at multiple locations were configured to automatically send clinical DICOM images to a DICOM receiver. A suite of custom MATLAB programs was established to extract and store public and private header data weekly. Specific use cases are provided for systematic image acquisition investigation, image processing harmonization, exposure index (EI) longitudinal monitoring and EI target optimization.ResultsFor systematic acquisition investigation, an example of adult lumbar spine exam analysis was provided with statistics on manual acquisition versus the use of automatic exposure control (AEC, including AEC dose level, active cell, and backup timer), grid usage, and collimation for various projections. For processing harmonization, up to 12.6% of protocols were revealed to have processing parameter differences in an example of a mobile radiography fleet. In addition, inconsistent use of a post‐acquisition image size function was also demonstrated, which resulted in anatomy size display variations. Bimonthly monitoring of median EI values showed expected trends, including changes after an AEC dose level adjustment for adult posterior‐anterior chest imaging on a scanner system. An example of adult axillary shoulder EI target refinement was shared using the median value, eμ, based on the lognormal EI data distribution after parsing down to acquisitions with appropriate techniques.ConclusionsThis analytics program enables systematic analysis of image acquisition and processing details. The information provides invaluable insights into real practice patterns, which can support data‐driven quality standardization and optimization.

Background: Arthroscopic debridement for osteochondritis dissecans (OCD) lesions of the capitellum is a relatively common and straightforward surgical option for failure of nonoperative management. However, the long-term outcomes of this procedure remain unknown. Hypothesis: Arthroscopic debridement of capitellar OCD would provide satisfactory long-term improvement in patient-reported outcomes. Study Design: Case series; Level of evidence, 4. Methods: Patients aged ≤18 years who underwent arthroscopic debridement procedures for OCD lesions (International Cartilage Repair Society grades 3 and 4) were identified. Procedures included loose body removal when needed and direct debridement of the lesion; marrow stimulation with drilling or microfracture was added at the discretion of each surgeon. The cohort consisted of 53 elbows. Patient evaluation included visual analog scale for pain; motion; subjective satisfaction; Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) scores; reoperation; and rate of return to sports. Results: At a mean 11 years of follow-up (range, 5-23 years), the median visual analog scale score for pain was 0, and 96% of patients reported being improved as compared with how they were before surgery. The mean ± SD QuickDASH score was 4 ± 9 points (range, 0-52 points), and 80% of patients returned to their sports of interest. The arc of motion significantly improved from 115°± 28° preoperatively to 130°± 17° at latest follow-up ( P = .026). Seven elbows (13%) required revision surgery for OCD lesions, resulting in high rates of overall survivorship free of revision surgery: 90% (95% CI, 80%-96%) at 5 years and 88% (95% CI, 76%-94%) at 10 years. At final follow-up, 7 all-cause reoperations were performed without revision surgery on the OCD lesion. Conclusion: Arthroscopic debridement of grade 3 or 4 OCD lesions of the capitellum produced satisfactory patient-reported outcomes in a majority of elbows, although a subset of patients experienced residual symptoms. The inherent selection bias of our cohort should be considered when applying these results to the overall population with OCD lesions, as we do not recommend this procedure for all patients.

AbstractBackgroundRNA‐seq experiments have traditionally been done with short‐read sequencing technologies that, by nature, collapse all RNA isoforms for a given gene into a single expression measurement—a major oversimplification of the underlying biology. Collapsing all RNA isoforms for a single gene severely limits our ability to characterize all RNA isoforms and determine their individual downstream functions. While computational approaches for assembling short reads into full transcripts exist, these methods are inherently structurally inaccurate, especially when compared to full‐length sequencing possible with long‐read sequencing technologies. In contrast, long‐read sequencing technologies can sequence entire RNA molecules, allowing researchers to accurately quantify expression for the complete set of RNA isoform species, including de novo RNA isoforms. Long‐read sequencing is especially well suited for discovering novel isoforms in the recently released telomere‐to‐telomere (T2T) human reference genome (CHM13). The new CHM13 genome assembly resolved highly homologous regions that are challenging to study with short‐reads. Here we sequenced post‐mortem human brain tissue with long‐reads and aligned them to CHM13 to explore novel gene bodies and transcript isoforms.MethodsWe sequenced pre‐frontal cortex tissue from four post‐mortem human brain samples using Oxford Nanopore Technologies long‐read sequencing for PCR amplified cDNA. Data were basecalled using Guppy, reads were aligned to the CHM13 human reference genome using minimap2, and transcripts were assembled and quantified with the Bambu package in R.ResultsAmong other findings, we discovered 84 new, high‐confidence gene bodies expressed in all four samples with at least 5 reads in each sample. We also found 223 novel RNA isoforms in previously annotated gene bodies. Of these 223 novel isoforms, 29 aligned to medically relevant genes such as MAOB, HLA‐DRB1, and ABO.ConclusionsOur results suggest long‐reads aligned to the CHM13 reference genome have the potential to reveal novel gene bodies and transcript isoforms that were missed in previous studies. These methods can provide a more complete picture of the transcriptomic landscape of diseases, including Alzheimer’s disease—hopefully generating information with potential to help inform future treatment and/or early diagnostic efforts.