Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

BACKGROUND: In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). HYPOTHESIS/OBJECTIVES: To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). ANIMALS: Ten healthy dogs, 21 dogs with PCC. METHODS: Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. RESULTS: Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Nivolumab Reaches Brain Lesions in Patients with Recurrent Glioblastoma and Induces T-cell Activity and Upregulation of Checkpoint Pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment.

Immunotherapy drives mesenchymal tumor cell state shift and TME immune response in glioblastoma patients.

BACKGROUND: Glioblastoma is a highly aggressive type of brain tumor for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. METHODS: Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. RESULTS: We present the integrative paired single-cell RNA-seq analysis of 76 tumor samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumor cells and the tumor microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumor cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumor cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (P = .0041). CONCLUSIONS: We find a resistance mechanism signature in one fifth of glioblastoma patients associated with a tumor-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumor cells.

Metronome-guided cochlear implantation for slower and smoother insertions of lateral wall electrodes.

INTRODUCTION: Achieving a slow and smooth electrode array insertion is paramount for preserving structural and functional integrity during cochlear implantation. This controlled study evaluates the efficacy of a metronome-guided insertion technique in enhancing the smoothness and speed of electrode array insertions. METHODS: In a prospective cohort study, patients undergoing cochlear implant surgery between 2022 and 2023 with lateral wall electrode arrays were included. Metronome guidance was delivered through an acoustic signal via headphones during electrode array insertion in cochlear implantation and compared to a control group without metronome-guidance. RESULTS: In total, 37 cases were evaluated, including 25 conventional insertions and 12 metronome-guided insertions. The results indicate that metronome-guided insertions were significantly slower (- 0.46 mm/s; p < 0.001) without extending the overall procedure time. This can be attributed to fewer paused sections observed in the metronome-guided technique. Moreover, metronome-guided insertions exhibited superior performance in terms of insertion smoothness and a reduced number of re-gripping events. CONCLUSIONS: The findings support the recommendation for the systematic application of metronome guidance in the manual insertion of cochlear implant electrode arrays, emphasizing its potential to optimize surgical outcomes.

Depression self-labeling in U.S. college students: Associations with perceived control and coping strategies.

BACKGROUND: Research on mental illness labeling has demonstrated that self-labeling (identifying with a mental illness label, e.g., "I have depression") is associated with internalized stigma, maladaptive responses to that stigma, and lower quality of life. However, research has not yet examined the link between self-labeling and how individuals cope with emotional distress. It is important to understand this relationship because adaptive and maladaptive methods of coping can lead to positive and negative mental illness outcomes. METHODS: This cross-sectional study examined the link between depression self-labeling, depression symptoms, and three constructs related to depression self-management (perceived control over depression, cognitive emotion regulation strategies, and help-seeking beliefs) in a large (N = 1423) sample of U.S. college students. RESULTS: Approximately one-fifth of students (22.2 %) self-labeled as having depression, while 39.0 % were estimated to meet diagnostic criteria for MDD. After controlling for depression symptom severity, self-labeling was associated with lower levels of perceived control over depression (p = .002), more catastrophizing (p = .013), less perspective taking, refocusing, reappraisal, and planning (ps < 0.05), and more positive help-seeking attitudes towards medication (p < .001) but not therapy. LIMITATIONS: Results are non-causal and may not generalize to non-college populations. CONCLUSIONS: Self-labeling may inform how individuals cope with emotional distress, with the potential for positive and negative effects on clinical outcomes. This is consistent with well-established research on self-labeling with regards to stigma, but extends this research in important new directions.

Prognostic Significance of the Microenvironment in Human Papillomavirus Oropharyngeal Carcinoma: A Systematic Review.

OBJECTIVE: The immune microenvironment of HPV-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) (HPV+OPSCCs) differs from that of HPV-independent oropharyngeal cancers (HPV-independent OPSCCs). The literature on the subject is very abundant, demanding an organized synthesis of this wealth of information to evaluate the hypothesis associating the favorable prognosis of HPV+OPSCC patients with a different immune microenvironment. A systematic review of the literature was conducted regarding the microenvironment of HPV+OPSCCs. DATA SOURCE: MEDLINE/PubMed, Embase, and Cochrane Library databases. REVIEW METHODS: A literature search was performed following PRISMA guidelines (Moher D. PLoS Med. 2009). The PEO (Population, Exposure, and Outcome) framework is detailed as follows: P: patients with oropharyngeal squamous cell carcinomas, E: human papillomavirus (HPV), and O: histological and immunological composition of the tumoral microenvironment (TME). No meta-analysis was performed. RESULTS: From 1,202 studies that were screened, 58 studies were included (n = 6,474 patients; n = 3,581 (55%) HPV+OPSCCs and n = 2,861(45%) HPV-independent OPSCCs). The presence of tumor-infiltrating lymphocytes (TIL), CD3+ in 1,733 patients, CD4+ in 520 patients, and CD8+ (cytotoxic T lymphocytes (CTL)) in 3,104 patients, and high levels of PD-L1 expression in 1,222 patients is strongly correlated with an improved clinical outcome in HPV+OPSCCs. CONCLUSION: This systematic review provides the most comprehensive information on the immune microenvironment of HPV+OPSCCs to date. Tumor-infiltrating lymphocytes and PD-L1 expression are associated with a favorable prognosis. B, CD8+ and resident memory cells densities are higher in HPV+OPSCCs. The importance of myeloid lineages is still a matter of debate and research. LEVEL OF EVIDENCE: NA Laryngoscope, 134:1507-1516, 2024.

Laparoscopic vs. open adrenalectomy: perioperative data and survival analysis in 70 dogs with an adrenal tumor.

Adrenalectomy is the treatment of choice in case of functional adrenal tumors and malignant adrenal incidentalomas. Laparoscopic adrenalectomy (LA) in dogs has gained popularity in recent years, however, clinical studies on large patient populations are scarce. This retrospective study describes perioperative and recurrence data, survival, and prognostic factors in 70 dogs that underwent LA or open adrenalectomy (OA) in our hospital between 2008 and 2022. Diagnosis was based on history, clinical signs, endocrine function tests and advanced diagnostic imaging. Laparoscopic adrenalectomy was performed in 42 dogs (n = 27 naturally occurring hypercortisolism, n = 4 pheochromocytoma, n = 1 pheochromocytoma with concurrent hypercortisolism, n = 10 incidentaloma) and OA in 28 dogs (n = 22 hypercortisolism, n = 3 pheochromocytoma, n = 3 incidentaloma). Bilateral adrenalectomy was performed in 8/70 dogs. Surgical duration of LA and OA did not differ significantly in unilateral and bilateral procedures (P = 0.108 and P = 0.101, respectively). Systemic hypertension occurred in 7/41 and 1/28 dogs during LA and OA, respectively (P = 0.130). Hypotension occurred in 2/41 and 4/28 dogs during LA and OA, respectively (P = 0.214). A total of 40/42 dogs in the LA group and 27/28 in the OA group survived to discharge (P = 0.810). Mean hospital stay was significantly shorter (P = 0.006) after LA (1.5 days, range 1-3) than after OA (2.2 days, range 1-4). No significant differences were demonstrated between LA and OA groups in recurrence of adrenal-dependent endocrine disease (P = 0.332), disease-free period (P = 0.733) and survival time (P = 0.353). The disease-specific 1-, 2- and 3-year survival rates were 95, 89, and 89% after LA and 92, 88, and 81% after OA. Tumor size was significantly associated with the occurrence of a recurrence. In addition, tumor size had a negative effect on the disease-free period and survival time. This study shows a favorable outcome of both LA and OA in dogs. Based on low perioperative complication rate, short hospitalization time and long-term outcomes comparable to OA in selected cases, the less invasive laparoscopic approach is considered the preferred technique.

Whole transcriptome analysis of canine pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies. To identify druggable pathways driving PPGL development, we performed RNA sequencing on PPGLs (n = 19) and normal adrenal medullas (NAMs; n = 10) of dogs. Principal component analysis (PCA) revealed that PPGLs clearly clustered apart from NAMs. In total, 4,218 genes were differentially expressed between PPGLs and NAMs. Of these, 232 had a log2 fold change of >3 or < -3, of which 149 were upregulated in PPGLs, and 83 were downregulated. Compared with NAMs, PPGLs had increased expression of genes related to the cell cycle, tumor development, progression and metastasis, hypoxia and angiogenesis, and the Wnt signaling pathway, and decreased expression of genes related to adrenal steroidogenesis. Our data revealed several overexpressed genes that could provide targets for novel therapeutics, such as Ret Proto-Oncogene (RET), Dopamine Receptor D2 (DRD2), and Secreted Frizzled Related Protein 2 (SFRP2). Based on the PCA, PPGLs were classified into 2 groups, of which group 1 had significantly higher Ki67 scores (p = 0.035) and shorter survival times (p = 0.04) than group 2. Increased expression of 1 of the differentially expressed genes between group 1 and 2, pleiotrophin (PTN), appeared to correlate with a more aggressive tumor phenotype. This study has shed light on the transcriptomic profile of canine PPGL, yielding new insights into the pathogenesis of these tumors in dogs, and revealed potential novel targets for therapy. In addition, we identified 2 transcriptionally distinct groups of PPGLs that had significantly different survival times.

Prevalence of persistent hypertension and situational hypertension in a population of elderly cats in The Netherlands.

OBJECTIVES: Systemic arterial hypertension is increasingly recognised and can have serious adverse consequences in cats. Unfortunately, the act of measuring blood pressure itself may cause an increase in blood pressure, known as situational hypertension. It is currently unknown how often this phenomenon occurs. The aim of this study was to evaluate the prevalence of persistent hypertension and situational hypertension in an elderly population of cats in a first-opinion clinic and to assess which factors were associated with systolic hypertension. METHODS: In this prospective study, systolic blood pressure was measured in 185 cats aged ⩾10 years using the Doppler sphygmomanometry method according to the recommendations of the American College of Veterinary Internal Medicine consensus statement. Age, sex, body weight, body condition score, position during blood pressure measurement and apparent stress level were assessed. If a systolic blood pressure >160 mmHg was found, measurements were repeated to evaluate if persistent hypertension or situational hypertension was present. The first set of blood pressure measurements were used for all the statistical analyses. RESULTS: The median systolic blood pressure for this population was 140 mmHg. The prevalence of persistent hypertension was at least 14.6% and situational hypertension at least 5.4%. Factors significantly associated with hypertension were age, higher apparent stress levels and a sitting position during measurement. Sex, body weight or body condition score did not significantly influence systolic blood pressure. CONCLUSIONS AND RELEVANCE: Both persistent hypertension and situational hypertension are common in elderly cats. There are no reliable parameters to distinguish between the two, underlining the importance of a standard protocol and repeating measurements during a follow-up visit when hypertension is found. Age, demeanour and body position during blood pressure measurement influenced blood pressure in this population of elderly cats.

Autologous dendritic cells loaded with antigens from self-renewing autologous tumor cells as patient-specific therapeutic cancer vaccines.

A promising personal immunotherapy is autologous dendritic cells (DC) loaded ex vivo with autologous tumor antigens (ATA) derived from self-renewing autologous cancer cells. DC-ATA are suspended in granulocyte-macrophage colony stimulating factor at the time of each subcutaneous injection. Previously, irradiated autologous tumor cell vaccines have produced encouraging results in 150 cancer patients, but the DC-ATA vaccine demonstrated superiority in single-arm and randomized trials in metastatic melanoma. DC-ATA have been injected into more than 200 patients with melanoma, glioblastoma, and ovarian, hepatocellular, and renal cell cancers. Key observations include: [1] greater than 95% success rates for tumor cell cultures and monocyte collection for dendritic cell production; [2] injections are well-tolerated; [3] the immune response is rapid and includes primarily TH1/TH17 cellular responses; [4] efficacy has been suggested by delayed but durable complete tumor regressions in patients with measurable disease, by progression-free survival in glioblastoma, and by overall survival in melanoma.

Reference intervals for plasma, urinary, and salivary concentrations of free metanephrines in dogs: Relevance to the diagnosis of pheochromocytoma.

BACKGROUND: Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs). HYPOTHESIS/OBJECTIVES: To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI). ANIMALS: Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs. METHODS: Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively. RESULTS: The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P < .001) and NAI (P = .002). The PCC dogs had significantly higher pMN than HC dogs (P < .001), but not higher than NAI dogs (P = .29). Using the upper reference limit, pNMN (>3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99). CONCLUSIONS AND CLINICAL IMPORTANCE: With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC.

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target.

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%-20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log2 fold change was >2 or < -2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans.

Phase 2 study of AV-GBM-1 (a tumor-initiating cell targeted dendritic cell vaccine) in newly diagnosed Glioblastoma patients: safety and efficacy assessment.

BACKGROUND: Vaccine immunotherapy may improve survival in Glioblastoma (GBM). A multicenter phase II trial was designed to determine: (1) the success rate of manufacturing the Aivita GBM vaccine (AV-GBM-1), (2) Adverse Events (AE) associated with AV-GBM-1 administration, and (3) survival. METHODS: Fresh suspected glioblastoma tissue was collected during surgery, and patients with pathology-confirmed GBM enrolled before starting concurrent Radiation Therapy and Temozolomide (RT/TMZ) with Intent to Treat (ITT) after recovery from RT/TMZ. AV-GBM-1 was made by incubating autologous dendritic cells with a lysate of irradiated autologous Tumor-Initiating Cells (TICs). Eligible patients were adults (18 to 70 years old) with a Karnofsky Performance Score (KPS) of 70 or greater, a successful TIC culture, and sufficient monocytes collected. A cryopreserved AV-GBM-1 dose was thawed and admixed with 500 µg of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) before every subcutaneous (s.c.) administration. RESULTS: Success rates were 97% for both TIC production and monocyte collection. AV-GBM-1 was manufactured for 63/63 patients; 60 enrolled per ITT; 57 started AV-GBM-1. The most common AEs attributed to AV-GBM-1 were local injection site reactions (16%) and flu-like symptoms (10%). Treatment-emergent AEs included seizures (33%), headache (37%), and focal neurologic symptoms (28%). One patient discontinued AV-GBM-1 because of seizures. Median Progression-Free Survival (mPFS) and median Overall Survival (mOS) from ITT enrollment were 10.4 and 16.0 months, respectively. 2-year Overall Survival (OS) is 27%. CONCLUSIONS: AV-GBM-1 was reliably manufactured. Treatment was well-tolerated, but there were numerous treatment-emergent central nervous system AEs. mPFS was longer than historical benchmarks, though no mOS improvement was noted. TRIAL REGISTRATION: NCT, NCT03400917 , Registered 10 January 2018.

Radiation Dosimetry of Theragnostic Pairs for Isotopes of Iodine in IAZA.

Theragnostic pairs of isotopes are used to infer radiation dosimetry for a therapeutic radiopharmaceutical from a diagnostic imaging study with the same tracer molecule labelled with an isotope better suited for the imaging task. We describe the transfer of radiation dosimetry from the diagnostic radioiodine isotope 123I, labelled for the hypoxia tracer molecule iodoazomycin arabinoside ([123I]IAZA), to isotopes 131I (therapeutic) and 124I (PET imaging). Uncertainties introduced by the dissimilar isotope half-lives are discussed in detail. Radioisotope dosimetries for [123I]IAZA were obtained previously. These data are used here to calculate residence times for 131I and 124I and their uncertainties. We distinguish two cases when extrapolating to infinity: purely physical decay (case A) and physical decay plus biological washout (case B). Organ doses were calculated using the MIRD schema with the OLIDNA/EXM code. Significant increases in some organ doses (in mSv per injected activity) were found for 131I and 124I. The most affected organs were the intestinal walls, thyroid, and urinary bladder wall. Uncertainty remained similar to 123I for case A but considerably greater for case B, especially for long biological half-lives (GI tract). Normal tissue dosimetries for IAZA must be considered carefully when substituting isotope species. A long biological half-life can significantly increase dosimetric uncertainties. These findings are relevant when considering PET imaging studies with [124I]IAZA or therapeutic administration of [131I]IAZA.

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

A personal COVID-19 dendritic cell vaccine made at point-of-care: Feasibility, safety, and antigen-specific cellular immune responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a world-wide pandemic. Internationally, because of availability, accessibility, and distribution issues, there is a need for additional vaccines. This study aimed to: establish the feasibility of personal dendritic cell vaccines to the SARS-CoV-2 spike protein, establish the safety of a single subcutaneous vaccine injection, and determine the antigen-specific immune response following vaccination. In Phase 1, 31 subjects were assigned to one of nine formulations of autologous dendritic cells and lymphocytes (DCL) incubated with 0.10, 0.33, or 1.0 µg of recombinant SARS-CoV-2 spike protein, and admixed with saline or 250 or 500 µg of granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to injection, then assessed for safety and humoral response. In Phase 2, 145 subjects were randomized to one of three formulations defined by incubation with the same three quantities of spike protein without GM-CSF, then assessed for safety and cellular response. Vaccines were successfully manufactured for every subject at point-of-care. Approximately 46.4% of subjects had a grade 1 adverse event (AE); 6.5% had a grade 2 AE. Among 169 evaluable subjects, there were no acute allergic, grade 3 or 4, or serious AE. In Phase 1, anti-receptor binding domain antibodies were increased in 70% of subjects on day-28. In Phase 2, in the 127 subjects who did not have high levels of gamma interferon-producing cells at baseline, 94.4% had increased by day 14 and 96.8% by day 28. Point-of-care personal vaccine manufacturing was feasible. Further development of such subject-specific vaccines is warranted.

Diagnostic yield of simultaneous dynamic contrast-enhanced magnetic resonance perfusion measurements and [18F]FET PET in patients with suspected recurrent anaplastic astrocytoma and glioblastoma.

PURPOSE: Both amino acid positron emission tomography (PET) and magnetic resonance imaging (MRI) blood volume (BV) measurements are used in suspected recurrent high-grade gliomas. We compared the separate and combined diagnostic yield of simultaneously acquired dynamic contrast-enhanced (DCE) perfusion MRI and O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET) PET in patients with anaplastic astrocytoma and glioblastoma following standard therapy. METHODS: A total of 76 lesions in 60 hybrid [18F]FET PET/MRI scans with DCE MRI from patients with suspected recurrence of anaplastic astrocytoma and glioblastoma were included retrospectively. BV was measured from DCE MRI employing a 2-compartment exchange model (2CXM). Diagnostic performances of maximal tumour-to-background [18F]FET uptake (TBRmax), maximal BV (BVmax) and normalised BVmax (nBVmax) were determined by ROC analysis using 6-month histopathological (n = 28) or clinical/radiographical follow-up (n = 48) as reference. Sensitivity and specificity at optimal cut-offs were determined separately for enhancing and non-enhancing lesions. RESULTS: In progressive lesions, all BV and [18F]FET metrics were higher than in non-progressive lesions. ROC analyses showed higher overall ROC AUCs for TBRmax than both BVmax and nBVmax in both lesion-wise (all lesions, p = 0.04) and in patient-wise analysis (p < 0.01). Combining TBRmax with BV metrics did not increase ROC AUC. Lesion-wise positive fraction/sensitivity/specificity at optimal cut-offs were 55%/91%/84% for TBRmax, 45%/77%/84% for BVmax and 59%/84%/72% for nBVmax. Combining TBRmax and best-performing BV cut-offs yielded lesion-wise sensitivity/specificity of 75/97%. The fraction of progressive lesions was 11% in concordant negative lesions, 33% in lesions only BV positive, 64% in lesions only [18F]FET positive and 97% in concordant positive lesions. CONCLUSION: The overall diagnostic accuracy of DCE BV imaging is good, but lower than that of [18F]FET PET. Adding DCE BV imaging did not improve the overall diagnostic accuracy of [18F]FET PET, but may improve specificity and allow better lesion-wise risk stratification than [18F]FET PET alone.

Circulating MicroRNAs as Non-invasive Biomarkers for Canine Cushing's Syndrome.

Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic (disease recurrence, histological grade) non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre- and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10-4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of <11. These results indicate that circulating miRNAs have the potential to be non-invasive biomarkers in dogs with Cushing's syndrome that may contribute to clinical decision making.

Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy.

Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats.

End-stage age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two major retinal degenerative (RD) conditions that result in irreversible vision loss. Permanent eye damage can also occur in battlefields or due to accidents. This suggests there is an unmet need for developing effective strategies for treating permanent retinal damages. In previous studies, co-grafted sheets of fetal retina with its retinal pigment epithelium (RPE) have demonstrated vision improvement in rat retinal disease models and in patients, but this has not yet been attempted with stem-cell derived tissue. Here we demonstrate a cellular therapy for irreversible retinal eye injuries using a "total retina patch" consisting of retinal photoreceptor progenitor sheets and healthy RPE cells on an artificial Bruch's membrane (BM). For this, retina organoids (ROs) (cultured in suspension) and polarized RPE sheets (cultured on an ultrathin parylene substrate) were made into a co-graft using bio-adhesives [gelatin, growth factor-reduced matrigel, and medium viscosity (MVG) alginate]. In vivo transplantation experiments were conducted in immunodeficient Royal College of Surgeons (RCS) rats at advanced stages of retinal degeneration. Structural reconstruction of the severely damaged retina was observed based on histological assessments and optical coherence tomography (OCT) imaging. Visual functional assessments were conducted by optokinetic behavioral testing and superior colliculus electrophysiology. Long-term survival of the co-graft in the rat subretinal space and improvement in visual function were observed. Immunohistochemistry showed that co-grafts grew, generated new photoreceptors and developed neuronal processes that were integrated into the host retina. This novel approach can be considered as a new therapy for complete replacement of a degenerated retina.