CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses.

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.

A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection.

Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Circulating noradrenaline leads to release of neuropeptide Y from cardiac sympathetic nerve terminals via activation of ß-adrenergic receptors.

Cardiac disease is marked by sympathoexcitation and elevated levels of noradrenaline (NA) and cotransmitter neuropeptide Y (NPY). Increased NPY levels are associated with a greater risk of ventricular arrhythmias and mortality. Nonetheless, the factors that cause NPY release remain poorly understood. We hypothesized that circulating catecholamines might lead to NPY release from myocardial sympathetic nerve terminals via a ß-receptor-mediated mechanism that enhances sympathoexcitation. Ventricular interstitial NA and NPY levels were measured in six Yorkshire pigs after i.v. administration of NA (1 mg) and before and after propranolol infusion (1 mg/kg). Real-time interstitial NPY levels were measured using ventricular capacitive immunoprobes (CIs) affixed with NPY antibodies and quantified as the change in CI input current (INPY ) upon binding of NPY. Interstitial NA was measured with adjacent fast-scan cyclic voltammetry probes (INA ). A left ventricular pressure catheter and continuous ECGs were used for haemodynamic recordings, and an epicardial 56-electrode sock was used for measurements of activation recovery interval, a surrogate of action potential duration. Upon administration of NA, heart rate and left ventricular pressure increased, and activation recovery interval shortened. Notably, NA significantly increased interstitial myocardial NPY levels. After propranolol, changes in heart rate and activation recovery interval were largely mitigated. The INA increased to a similar extent post-propranolol vs. pre-propranolol, but changes in INPY were significantly reduced post-propranolol. Coronary sinus plasma analyses confirmed fast-scan cyclic voltammetry and CI findings. Hence, this study demonstrates that circulating NA induces NPY release from ventricular sympathetic nerve terminals, the mechanism for which is mediated via ß-adrenergic receptors and can be blocked by the non-selective ß-blocker, propranolol. KEY POINTS: Cardiovascular disease is characterized by sympathovagal imbalance, with increased plasma noradrenaline (NA) and neuropeptide Y (NPY) concentrations. Increased NPY levels are associated with increased ventricular arrhythmias and mortality in heart failure. Limited data are available on the specific factors that cause NPY release. In this study, fast-scan cyclic voltammetry and capacitive immunoprobes were used to allow for real-time in vivo measurements of interstitial myocardial neurotransmitters and neuropeptides, respectively. Using an in vivo porcine model with cardiac fast-scan cyclic voltammetry and capacitive immunoprobes, it was shown that systemic NA can increase ventricular interstitial NPY levels, suggesting that NA induces NPY release from postganglionic sympathetic nerves. The release of NPY was blocked by administration of the non-selective ß-blocker propranolol, suggesting that release of NPY is dependent on activation of ß-adrenergic receptors by NA.

SARS-CoV-2-specific T cells generated for adoptive immunotherapy are capable of recognizing multiple SARS-CoV-2 variants.

Adoptive T-cell immunotherapy has provided promising results in the treatment of viral complications in humans, particularly in the context of immunocompromised patients who have exhausted all other clinical options. The capacity to expand T cells from healthy immune individuals is providing a new approach to anti-viral immunotherapy, offering rapid off-the-shelf treatment with tailor-made human leukocyte antigen (HLA)-matched T cells. While most of this research has focused on the treatment of latent viral infections, emerging evidence that SARS-CoV-2-specific T cells play an important role in protection against COVID-19 suggests that the transfer of HLA-matched allogeneic off-the-shelf virus-specific T cells could provide a treatment option for patients with active COVID-19 or at risk of developing COVID-19. We initially screened 60 convalescent individuals and based on HLA typing and T-cell response profile, 12 individuals were selected for the development of a SARS-CoV-2-specific T-cell bank. We demonstrate that these T cells are specific for up to four SARS-CoV-2 antigens presented by a broad range of both HLA class I and class II alleles. These T cells show consistent functional and phenotypic properties, display cytotoxic potential against HLA-matched targets and can recognize HLA-matched cells infected with different SARS-CoV-2 variants. These observations demonstrate a robust approach for the production of SARS-CoV-2-specific T cells and provide the impetus for the development of a T-cell repository for clinical assessment.

Risk Factors for Early Lower Limb Re-Amputation in Vascular Diseases.

BACKGROUND: Numerous risk factors for lower limb amputations are known; however, this study aimed to identify risk factors for re-amputation in patients within 6 months from an initial lower limb amputation procedure. METHODS: This single-center retrospective cohort study was performed at the Hospital Regional Hans Dieter Schmidt in Brazil. The study included patients who were aged at least 18 years and had undergone lower limb amputation between 2013 and 2022. Patients who died while hospitalized and patients who were lost to follow-up after hospital discharge were excluded from the study. Patient age, sex, number of amputations, revision time, comorbidities, and potential risk factors were extracted from the physical therapy service database and electronic medical records of the hospital. Chi-squared test and student's t-test were used to identify statistical significance. RESULTS: A total of 652 patients were included, of which 35.2% (230) patients underwent re-amputation within 6 months of the first operation. We found that dialysis (P = 0.004; odds ratio [OR] 8.36, 95% confidence interval [CI] 3.09-20.5), smoking (P = 0.004; OR 1.67, 95% CI 1.18-2.35), and hypertension (P = 0.02; OR 1.55, 95% CI 1.09-2.19) were predictive factors for re-amputation within 6 months of lower limb amputation. CONCLUSIONS: Therefore, it is important to intervene early and provide additional support to patients undergoing lower limb amputation with these risk factors to reduce the potential for re-amputation in the future.

Voltammetry in the spleen assesses real-time immunomodulatory norepinephrine release elicited by autonomic neurostimulation.

BACKGROUND: The noradrenergic innervation of the spleen is implicated in the autonomic control of inflammation and has been the target of neurostimulation therapies for inflammatory diseases. However, there is no real-time marker of its successful activation, which hinders the development of anti-inflammatory neurostimulation therapies and mechanistic studies in anti-inflammatory neural circuits. METHODS: In mice, we performed fast-scan cyclic voltammetry (FSCV) in the spleen during intravenous injections of norepinephrine (NE), and during stimulation of the vagus, splanchnic, or splenic nerves. We defined the stimulus-elicited charge generated at the oxidation potential for NE (~ 0.88 V) as the "NE voltammetry signal" and quantified the dependence of the signal on NE dose and intensity of neurostimulation. We correlated the NE voltammetry signal with the anti-inflammatory effect of splenic nerve stimulation (SpNS) in a model of lipopolysaccharide- (LPS) induced endotoxemia, quantified as suppression of TNF release. RESULTS: The NE voltammetry signal is proportional to the estimated peak NE blood concentration, with 0.1 µg/mL detection threshold. In response to SpNS, the signal increases within seconds, returns to baseline minutes later, and is blocked by interventions that deplete NE or inhibit NE release. The signal is elicited by efferent, but not afferent, electrical or optogenetic vagus nerve stimulation, and by splanchnic nerve stimulation. The magnitude of the signal during SpNS is inversely correlated with subsequent TNF suppression in endotoxemia and explains 40% of the variance in TNF measurements. CONCLUSIONS: FSCV in the spleen provides a marker for real-time monitoring of anti-inflammatory activation of the splenic innervation during autonomic stimulation.

Interfascicular Anatomy of the Motor Branch of the Ulnar Nerve: A Cadaveric Study.

PURPOSE: The motor branch of the ulnar nerve contains fascicles that innervate the intrinsic musculature of the hand. This cadaveric study aimed to describe the organization and consistency of the internal topography of the motor branch of the ulnar nerve. METHODS: Five fresh-frozen cadaveric specimens with an average age of 74 years (range, 65-88 years) were dissected. The ulnar nerve was exposed and transfixed to the underlying tissues to maintain its orientation throughout the dissection. The dorsal cutaneous branch (DCB) and the volar sensory branch were identified and reflected to expose the motor branch. The fascicles to the first dorsal interosseus (FDI), flexor pollicis brevis, and abductor digiti minimi (ADM) were identified. Internal neurolysis was performed distal to proximal to identify the interfascicular arrangement of these fascicles within the motor branch. The organization of these fascicles was noted, and the branch points of the DCB, FDI, and ADM were measured relative to the pisiform using a handheld electronic caliper. RESULTS: The internal topography of the motor branch was consistent among all specimens. Proximal to the pisiform, the arrangement from radial to ulnar was as follows: volar sensory branch, flexor pollicis brevis, FDI/intrinsic muscles, ADM, and DCB. The position of these branches remained consistent as the deep motor branch curved radially within the palm and traveled to the terminal musculature. The locations of the average branch points of the FDI, ADM, and DCB with respect to the pisiform were as follows: FDI, 4.6 cm distal (range, 4.1-4.9 cm), 4.5 cm radial (range, 4.1-4.9 cm); ADM, 0.65 cm distal (range, 0.3-1.1 cm), 0.7 cm radial (range, 0.3-1.1 cm), DCB, 7.7 cm proximal (range, 4.2-10.1 cm), and 0.4 cm ulnar (range, 0.3-0.8 cm). CONCLUSIONS: The internal topography of the ulnar nerve motor branch was consistent among the specimens studied. The topography of the motor branches was maintained as the motor branch turns radially within the palm. CLINICAL RELEVANCE: This study provides further understanding of the internal topography of the ulnar nerve motor branch at the wrist level.

Robot-Assisted Nasal Reconstruction: A Cadaveric Study.

OBJECTIVE: Manual contouring of cartilage for nasal reconstruction is tedious and time-consuming. The use of a robot could improve the speed and precision of the contouring process. This cadaveric study evaluates the efficiency and accuracy of a robot methodology for contouring the lower lateral cartilage of the nasal tip. METHODS: An augmented robot with a spherical burring tool attached was utilized to carve 11 cadaveric rib cartilage specimens. In phase 1, the right lower lateral cartilage was harvested from a cadaveric specimen and used to define a carving path for each rib specimen. In phase 2, the cartilage remained in situ during the scanning and 3-dimensional modeling. The final carved specimens were compared with the preoperative plans through topographical accuracy analysis. The contouring times of the specimens were compared with 14 retrospectively reviewed cases (2017-2020) by an experienced surgeon. RESULTS: Phase 1 root mean square error of 0.40±0.15 mm and mean absolute deviation of 0.33±0.13 mm. Phase 2 root mean square error of 0.43 mm and mean absolute deviation of 0.28 mm. The average carving time for the robot specimens was 14±3 minutes and 16 minutes for Phase 1 and Phase 2, respectively. The average manual carving by an experienced surgeon was 22±4 minutes. CONCLUSIONS: Robot-assisted nasal reconstruction is very precise and more efficient than manual contouring. This technique represents an exciting and innovative alternative for complex nasal reconstruction.

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein.

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

Scalable and reversible axonal neuromodulation of the sympathetic chain for cardiac control.

Maladaptation of the sympathetic nervous system contributes to the progression of cardiovascular disease and risk for sudden cardiac death, the leading cause of mortality worldwide. Axonal modulation therapy (AMT) directed at the paravertebral chain blocks sympathetic efferent outflow to the heart and maybe a promising strategy to mitigate excess disease-associated sympathoexcitation. The present work evaluates AMT, directed at the sympathetic chain, in blocking sympathoexcitation using a porcine model. In anesthetized porcine (n = 14), we applied AMT to the right T1-T2 paravertebral chain and performed electrical stimulation of the distal portion of the right sympathetic chain (RSS). RSS-evoked changes in heart rate, contractility, ventricular activation recovery interval (ARI), and norepinephrine release were examined with and without kilohertz frequency alternating current block (KHFAC). To evaluate efficacy of AMT in the setting of sympathectomy, evaluations were performed in the intact state and repeated after left and bilateral sympathectomy. We found strong correlations between AMT intensity and block of sympathetic stimulation-evoked changes in cardiac electrical and mechanical indices (r = 0.83-0.96, effect size d = 1.9-5.7), as well as evidence of sustainability and memory. AMT significantly reduced RSS-evoked left ventricular interstitial norepinephrine release, as well as coronary sinus norepinephrine levels. Moreover, AMT remained efficacious following removal of the left sympathetic chain, with similar mitigation of evoked cardiac changes and reduction of catecholamine release. With growth of neuromodulation, an on-demand or reactionary system for reversible AMT may have therapeutic potential for cardiovascular disease-associated sympathoexcitation.NEW & NOTEWORTHY Autonomic imbalance and excess sympathetic activity have been implicated in the pathogenesis of cardiovascular disease and are targets for existing medical therapy. Neuromodulation may allow for control of sympathetic projections to the heart in an on-demand and reversible manner. This study provides proof-of-concept evidence that axonal modulation therapy (AMT) blocks sympathoexcitation by defining scalability, sustainability, and memory properties of AMT. Moreover, AMT directly reduces release of myocardial norepinephrine, a mediator of arrhythmias and heart failure.

Protocol for SARS-CoV-2 post-vaccine surveillance study in Australian adults and children with cancer: an observational study of safety and serological and immunological response to SARS-CoV-2 vaccination (SerOzNET).

BACKGROUND: Cancer is associated with excess morbidity and mortality from coronavirus disease 2019 (COVID-19) following infection by the novel pandemic coronavirus SARS-CoV-2. Vaccinations against SARS-CoV-2 have been rapidly developed and proved highly effective in reducing the incidence of severe COVID-19 in clinical trials of healthy populations. However, patients with cancer were excluded from pivotal clinical trials. Early data suggest that vaccine response is less robust in patients with immunosuppressive conditions or treatments, while toxicity and acceptability of COVID-19 vaccines in the cancer population is unknown. Unanswered questions remain about the impact of various cancer characteristics (such as treatment modality and degree of immunosuppression) on serological response to and safety of COVID-19 vaccinations. Furthermore, as the virus and disease manifestations evolve, ongoing data is required to address the impact of new variants. METHODS: SerOzNET is a prospective observational study of adults and children with cancer undergoing routine SARS-CoV-2 vaccination in Australia. Peripheral blood will be collected and processed at five timepoints (one pre-vaccination and four post-vaccination) for analysis of serologic responses to vaccine and exploration of T-cell immune correlates. Cohorts include: solid organ cancer (SOC) or haematological malignancy (HM) patients currently receiving (1) chemotherapy, (2) immune checkpoint inhibitors (3) hormonal or targeted therapy; (4) patients who completed chemotherapy within 6-12 months of vaccination; (5) HM patients with conditions associated with hypogammaglobulinaemia or immunocompromise; (6) SOC or HM patients with allergy to PEG or polysorbate 80. Data from healthy controls already enrolled on several parallel studies with comparable time points will be used for comparison. For children, patients with current or prior cancer who have not received recent systemic therapy will act as controls. Standardised scales for quality-of-life assessment, patient-reported toxicity and vaccine hesitancy will be obtained. DISCUSSION: The SerOzNET study was commenced in June 2021 to prospectively study immune correlates of vaccination in specific cancer cohorts. The high proportion of the Australian population naïve to COVID-19 infection and vaccination at study commencement has allowed a unique window of opportunity to study vaccine-related immunity. Quality of life and patient-reported adverse events have not yet been reported in detail post-vaccination for cancer patients. Trial registration This trial is registered on the Australia New Zealand Clinical Trials Registry (ANZCTR) ACTRN12621001004853. Submitted for registration 25 June 2021. Registered 30 July 2021 (Retrospectively registered). https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382281&isReview=true.

Pretransplant Cytomegalovirus-Specific Cellular Immunity and Risk of Viral Reactivation Following Lung Transplantation: A Prospective Cohort Study.

Cytomegalovirus (CMV) remains a significant burden in lung transplant recipients. Deficiencies in T-cell immunity posttransplant increase the risk of CMV-associated complications. However, it is not clear if underlying poor pretransplant immunity increases risk. To assess this, we recruited 39 prospective lung transplant patients and performed QuantiFERON-CMV on their peripheral blood. More than a third of prospective CMV-seropositive transplant recipients were CMV non-immune reactive (CMV-NIR) pretransplant. CMV-NIR status was associated with a significantly higher incidence of CMV reactivation posttransplant, demonstrating that dysfunctional CMV immunity in prospective lung transplant recipients is associated with an increased risk of viral reactivation posttransplant.

Rapid measurement of cardiac neuropeptide dynamics by capacitive immunoprobe in the porcine heart.

Sympathetic control of regional cardiac function occurs through postganglionic innervation from stellate ganglia and thoracic sympathetic chain. Whereas norepinephrine (NE) is their primary neurotransmitter, neuropeptide Y (NPY) is an abundant cardiac cotransmitter. NPY plays a vital role in homeostatic processes including angiogenesis, vasoconstriction, and cardiac remodeling. Elevated sympathetic stress, resulting in increased NE and NPY release, has been implicated in the pathogenesis of several cardiovascular disorders including hypertension, myocardial infarction, heart failure, and arrhythmias, which may result in sudden cardiac death. Current methods for the detection of NPY in myocardium are limited in their spatial and temporal resolution and take days to weeks to provide results [e.g., interstitial microdialysis with subsequent analysis by enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), or mass spectrometry]. In this study, we report a novel approach for measurement of interstitial and intravascular NPY using a minimally invasive capacitive immunoprobe (C.I. probe). The first high-spatial and temporal resolution, multichannel measurements of NPY release in vivo are provided in both myocardium and transcardiac vascular space in a beating porcine heart. We provide NPY responses evoked by sympathetic stimulation and ectopic ventricular pacing and compare these to NE release and hemodynamic responses. We extend this approach to measure both NPY and vasoactive intestinal peptide (VIP) and show differential release profiles under sympathetic stimulation. Our data demonstrate rapid and local changes in neurotransmitter profiles in response to sympathetic cardiac stressors. Future implementations include real-time intraoperative determination of cardiac neuropeptides and deployment as a minimally invasive catheter.NEW & NOTEWORTHY The sympathetic nervous system regulates cardiac function through release of neurotransmitters and neuropeptides within the myocardium. Neuropeptide Y (NPY) acts as an acute cardiac vasoconstrictor and chronically to regulate angiogenesis and cardiac remodeling. Current methodologies for the measure of NPY are not capable of providing rapid readouts on a single-sample basis. Here we provide the first in vivo methodology to report dynamic, localized NPY levels within both myocardium and vascular compartments in a beating heart.

Building capacity through integration of advanced practice nurses in research.

BACKGROUND: Advanced practice nurses (APNs) are well-positioned to function in research settings, however barriers to their engagement persist. Capacity-building through multisite research opportunities is an important strategy to overcome these barriers. PURPOSE: To describe the benefits and challenges of incorporating APNs in research and discuss opportunities for building capacity for nursing research. METHOD: Grounded in the experience of a nurse-led multisite longitudinal observational descriptive symptom study, field notes representing the research continuum were reviewed and categorized into themes reflecting benefits and challenges. FINDINGS: Uniform benefits of acquiring research knowledge and skills, participating in research activities, and engaging in professional development were experienced among APNs. Limited support for regulatory and research activities, inadequate financial infrastructure, and a perceived lack of value for APNs' professional growth were commonly encountered challenges. DISCUSSION: Establishment of an infrastructure that elevates benefits and mitigates challenges is necessary to effectively incorporate APNs in a research environment, build capacity, and advance nursing science.

Fast in vivo detection of myocardial norepinephrine levels in the beating porcine heart.

The sympathetic nervous system modulates cardiac function by controlling key parameters such as chronotropy and inotropy. Sympathetic control of ventricular function occurs through extrinsic innervation arising from the stellate ganglia and thoracic sympathetic chain. In the healthy heart, sympathetic release of norepinephrine (NE) results in positive modulation of chronotropy, inotropy, and dromotropy, significantly increasing cardiac output. However, in the setting of myocardial infarction or injury, sympathetic activation persists, contributing to heart failure and increasing the risk of arrhythmias, including sudden cardiac death. Methodologies for detection of norepinephrine in cardiac tissue are limited. Present techniques rely on microdialysis for analysis by high-performance liquid chromatography coupled to electrochemical detection (HPLC-ED), radioimmunoassay, or other immunoassays, such as enzyme-linked immunosorbent assay (ELISA). Although significant information about the release and action of norepinephrine has been obtained with these methodologies, they are limited in temporal resolution, require large sample volumes, and provide results with a significant delay after sample collection (hours to weeks). In this study, we report a novel approach for measurement of interstitial cardiac norepinephrine, using minimally invasive, electrode-based, fast-scanning cyclic voltammetry (FSCV) applied in a beating porcine heart. The first multispatial and high temporal resolution, multichannel measurements of NE release in vivo are provided. Our data demonstrate rapid changes in interstitial NE profiles with regional differences in response to coronary ischemia, sympathetic nerve stimulation, and alterations in preload/afterload.NEW & NOTEWORTHY Pharmacological, electrical, or surgical regulation of sympathetic neuronal control can be used to modulate cardiac function and treat arrhythmias. However, present methods for monitoring sympathetic release of norepinephrine in the heart are limited in spatial and temporal resolution. Here, we provide for the first time a methodology and demonstration of practice and rapid measures of individualized regional autonomic neurotransmitter levels in a beating heart. We show dynamic, spatially resolved release profiles under normal and pathological conditions.

Colocalization of optical coherence tomography angiography with histology in the mouse retina.

Optical coherence tomography angiography (OCT-A) allows in vivo, non-invasive, functional imaging of retinal perfusion. The purpose of this study was to determine the reliability of OCT-A in visualizing the complete retinal vasculature by comparing in vivo OCT-A images to matched ex vivo retinal tissue in mice. Adult female C57BL/6 mice were imaged to obtain OCT-A images of the superficial vascular complex, intermediate capillary plexus and deep capillary plexus. Z-stack fluorescence images of whole-mounted retinas, labeled for vascular endothelial cells by anti-isolectin immunohistochemistry and FITC-dextran perfusion, were generated. The OCT-A and fluorescence images were manually colocalized and vessel length measured for each of the techniques. Mean vessel length among all plexuses showed less than 13% difference between OCT-A and lectin immunohistochemistry and less than 4% difference between OCT-A and FITC-dextran perfusion. The strength of the correlation between OCT-A and lectin immunohistochemistry ranged from 0.46-0.95, while that between OCT-A and FITC-perfusion ranged from 0.67-0.88. OCT-A visualized retinal vasculature in vivo to a similar extent in matched ex vivo histology images. Our results show that OCT-A is a reliable method for acquiring in vivo images of retinal perfusion in mice, with the ability to differentiate each vascular plexus.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

T-cell adoptive immunotherapy for BK nephropathy in renal transplantation.

INTRODUCTION: BK virus (BKPyV) nephropathy occurs in 1%-10% of kidney transplant recipients, with suboptimal therapeutic options. CASE: A 54-year-old woman received a transplant in March 2017. BKPyV was detected at 1.5 × 102  copies/mL within a month, necessitating halving of mycophenolate and addition of leflunomide. Allograft histology in December showed polyomavirus nephropathy treated with intravenous immunoglobulin and cessation of mycophenolate. In February 2018, cidofovir and ciprofloxacin were commenced. In April, tacrolimus was reduced while introducing everolimus. A second graft biopsy in August showed increasing polyoma virus infection and a subsequent biopsy in September for worsening renal function showed 30% of tubular reactivity for simian virus 40 (SV40). Allogeneic BKPyV-reactive T cells were generated from the patient's daughter and infused over 10 sessions starting late September. The fourth allograft biopsy in November 2018 demonstrated involvement of BKPyV in 50% of tubules. Allograft function continued to decline, requiring hemodialysis from December 2018. Allograft nephrectomy after 6 months showed <1% SV40 in preserved tubules and 80% interstitial fibrosis. DISCUSSION: We conclude that the T-cell adoptive immunotherapy reduced BKPyV load significantly despite extensive infection, but attendant fibrosis and tubular atrophy led to graft failure. Early intervention with T-cell therapy may prove efficacious in BKPyV nephropathy.

Autologous Adoptive T-cell Therapy for Recurrent or Drug-resistant Cytomegalovirus Complications in Solid Organ Transplant Recipients: A Single-arm Open-label Phase I Clinical Trial.

Background: Opportunistic infections including cytomegalovirus (CMV) are a major cause of morbidity and mortality in solid organ transplant (SOT) recipients. The recurrent and protracted use of antiviral drugs with eventual emergence of drug resistance represents a significant constraint to therapy. Although adoptive T-cell therapy has been successfully used in hematopoietic stem cell transplant recipients, its extension to the SOT setting poses a considerable challenge because of the inhibitory effects of immunosuppressive drugs on the virus-specific T-cell response in vivo and the perceived risk of graft rejection. Methods: In this prospective study, 22 SOT recipients (13 renal and 8 lung and 1 heart transplants) with recurrent or ganciclovir-resistant CMV infection were recruited, and 13 of them were treated with in vitro-expanded autologous CMV-specific T cells. These patients were monitored for safety, clinical symptoms, and immune reconstitution. Results: Autologous CMV-specific T-cell manufacture was attempted for 21 patients, and was successful in 20. The use of this adoptive immunotherapy was associated with no therapy-related serious adverse events. Eleven (84%) of the 13 treated patients showed improvement in symptoms, including complete resolution or reduction in DNAemia and CMV-associated end-organ disease and/or the cessation or reduced use of antiviral drugs. Furthermore, four of these patients showed coincident increased frequency of CMV-specific T cells in peripheral blood after completion of T-cell therapy. Conclusions: The data presented here demonstrate for the first time the clinical safety of CMV-specific adoptive T-cell therapy and its potential therapeutic benefit for SOT recipients with recurrent and/or drug-resistant CMV infection or disease. Clinical Trials Registration: ACTRN12613000981729.