Diffuse optical tomography for mapping cerebral hemodynamics and functional connectivity in delirium.

INTRODUCTION: Delirium is associated with mortality and new onset dementia, yet the underlying pathophysiology remains poorly understood. Development of imaging biomarkers has been difficult given the challenging nature of imaging delirious patients. Diffuse optical tomography (DOT) offers a promising approach for investigating delirium given its portability and three-dimensional capabilities. METHODS: Twenty-five delirious and matched non-delirious patients (n = 50) were examined using DOT, comparing cerebral oxygenation and functional connectivity in the prefrontal cortex during and after an episode of delirium. RESULTS: Total hemoglobin values were significantly decreased in the delirium group, even after delirium resolution. Functional connectivity between the dorsolateral prefrontal cortex and dorsomedial prefrontal cortex was strengthened post-resolution compared to during an episode; however, this relationship was still significantly weaker compared to controls. DISCUSSION: These findings highlight DOT's potential as an imaging biomarker to measure impaired cerebral oxygenation and functional dysconnectivity during and after delirium. Future studies should focus on the role of cerebral oxygenation in delirium pathogenesis and exploring the etiological link between delirium and dementias. HIGHLIGHTS: We developed a portable diffuse optical tomography (DOT) system for bedside three-dimensional functional neuroimaging to study delirium in the hospital. We implemented a novel DOT task-focused seed-based correlation analysis. DOT revealed decreased cerebral oxygenation and functional connectivity strength in the delirium group, even after resolution of delirium.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer.

BACKGROUND: Natural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. METHODS: Starting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach. RESULTS: Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. CONCLUSIONS: Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

Low-Dose Sorafenib Promotes Cancer Stem Cell Expansion and Accelerated Tumor Progression in Soft Tissue Sarcomas.

The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients.

Risk of adverse perinatal outcomes in pregnancies with "small" fetuses not meeting Delphi consensus criteria for fetal growth restriction.

BACKGROUND: Previous research endeavors examining the association between clinical characteristics, sonographic indices, and the risk of adverse perinatal outcomes in pregnancies complicated by fetal growth restriction have been hampered by a lack of agreement regarding its definition. In 2016, a consensus definition was reached by an international panel of experts via the Delphi procedure, but as it currently stands, this has not been endorsed by all professional organizations. OBJECTIVE: This study aimed to assess whether an independent association exists between estimated fetal weight and/or abdominal circumference of <10th percentile and adverse perinatal outcomes when consensus criteria for growth restriction are not met. STUDY DESIGN: Data were derived from a passive prospective cohort of singleton nonanomalous pregnancies at a single academic tertiary care institution (2010-2022) that fell into 3 groups: (1) consecutive fetuses that met the Delphi criteria for fetal growth restriction, (2) small-for-gestational-age fetuses that failed to meet the consensus criteria, and (3) fetuses with birthweights of 20th to 80th percentile randomly selected as an appropriately grown (appropriate-for-gestational-age) comparator group. This nested case-control study used 1:1 propensity score matching to adjust for confounders among the 3 groups: fetal growth restriction cases, small-for-gestational-age cases, and controls. Our primary outcome was a composite: perinatal demise, 5-minute Apgar score of <7, cord pH of ≤7.10, or base excess of ≥12. Pregnancy characteristics with a P value of <.2 on univariate analyses were considered for incorporation into a multivariable model along with fetal growth restriction and small-for-gestational-age to evaluate which outcomes were independently predictive of adverse perinatal outcomes. RESULTS: Overall, 2866 pregnancies met the inclusion criteria. After propensity score matching, there were 2186 matched pairs, including 511 (23%), 1093 (50%), and 582 (27%) patients in the small-for-gestational-age, appropriate-for-gestational-age, and fetal growth restriction groups, respectively. Moreover, 210 pregnancies (10%) were complicated by adverse perinatal outcomes. None of the pregnancies with small-for-gestational-age OR appropriate-for-gestational-age fetuses resulted in perinatal demise. Twenty-three of 511 patients (5%) in the small-for-gestational-age group had adverse outcomes based on 5-minute Apgar scores and/or cord gas results compared with 77 of 1093 patients (7%) in the appropriate-for-gestational-age group (odds ratio, 0.62; 95% confidence interval, 0.39-1.00). Furthermore, 110 of 582 patients (19%) with fetal growth restriction that met the consensus criteria had adverse outcomes (odds ratio, 3.08; 95% confidence interval, 2.25-4.20), including 34 patients with perinatal demise or death before discharge. Factors independently associated with increased odds of adverse outcomes included chronic hypertension, hypertensive disorders of pregnancy, and early-onset fetal growth restriction. Small-for-gestational age was not associated with the primary outcome after adjustment for 6 other factors included in a model predicting adverse perinatal outcomes. The bias-corrected bootstrapped area under the receiver operating characteristic curve for the model was 0.72 (95% confidence interval, 0.66-0.74). The bias-corrected bootstrapped area under the receiver operating characteristic curve for a 7-factor model predicting adverse perinatal outcomes was 0.72 (95% confidence interval, 0.66-0.74). CONCLUSION: This study found no evidence that fetuses with an estimated fetal weight and/or abdominal circumference of 3rd to 9th percentile that fail to meet the consensus criteria for fetal growth restriction (based on Doppler waveforms and/or growth velocity of ≥32 weeks) are at increased risk of adverse outcomes. Although the growth of these fetuses should be monitored closely to rule out evolving growth restriction, most cases are healthy constitutionally small fetuses. The management of these fetuses in the same manner as those with suspected pathologic growth restriction may result in unnecessary antenatal testing and increase the risk of iatrogenic complications resulting from preterm or early term delivery of small fetuses that are at relatively low risk of adverse perinatal outcomes.

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Experimental design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Results: Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

Surgical Management of Emphysematous Gastritis in a Postpartum Female: A Case Report and Literature Review.

Emphysematous gastritis is a form of gastritis characterized by both gastric inflammation and the presence of intramural gas. Its occurrence is rare, and its presentation is non-specific. Consequently, no definitive guidelines for management have been outlined. We herein detail the diagnosis and surgical management of a female with complicated emphysematous gastritis following a cesarean section delivery. In light of the gastric ischemia noted on esophagogastroduodenoscopy, the decision was made to proceed with surgical management to ascertain the extent of necrosis. Following a partial gastrectomy, the patient had an uneventful postoperative course and met feeding milestones. Given the high morbidity rate of emphysematous gastritis and the success of our intervention, we propose the utilization of exploratory laparoscopy/laparotomy in patients with identified or highly suspected gastric ischemia. This aids in the characterization of ischemia and guides decision-making on the extent of gastric resection (partial versus complete gastrectomy).

Inhaled recombinant human IL-15 in dogs with naturally occurring pulmonary metastases from osteosarcoma or melanoma: a phase 1 study of clinical activity and correlates of response.

PURPOSE: Although recombinant human interleukin-15 (rhIL-15) has generated much excitement as an immunotherapeutic agent for cancer, activity in human clinical trials has been modest to date, in part due to the risks of toxicity with significant dose escalation. Since pulmonary metastases are a major site of distant failure in human and dog cancers, we sought to investigate inhaled rhIL-15 in dogs with naturally occurring lung metastases from osteosarcoma (OSA) or melanoma. We hypothesized a favorable benefit/risk profile given the concentrated delivery to the lungs with decreased systemic exposure. EXPERIMENTAL DESIGN: We performed a phase I trial of inhaled rhIL-15 in dogs with gross pulmonary metastases using a traditional 3+3 cohort design. A starting dose of 10 µg twice daily × 14 days was used based on human, non-human primate, and murine studies. Safety, dose-limiting toxicities (DLT), and maximum tolerated dose (MTD) were the primary objectives, while response rates, progression-free and overall survival (OS), and pharmacokinetic and immune correlative analyses were secondary. RESULTS: From October 2018 to December 2020, we enrolled 21 dogs with 18 dogs reaching the 28-day response assessment to be evaluable. At dose level 5 (70 µg), we observed two DLTs, thereby establishing 50 µg twice daily × 14 days as the MTD and recommended phase 2 dose. Among 18 evaluable dogs, we observed one complete response >1 year, one partial response with resolution of multiple target lesions, and five stable disease for an overall clinical benefit rate of 39%. Plasma rhIL-15 quantitation revealed detectable and sustained rhIL-15 concentrations between 1-hour and 6 hour postnebulization. Decreased pretreatment lymphocyte counts were significantly associated with clinical benefit. Cytotoxicity assays of banked peripheral blood mononuclear cells revealed significant increases in peak cytotoxicity against canine melanoma and OSA targets that correlated with OS. CONCLUSIONS: In this first-in-dog clinical trial of inhaled rhIL-15 in dogs with advanced metastatic disease, we observed promising clinical activity when administered as a monotherapy for only 14 days. These data have significant clinical and biological implications for both dogs and humans with refractory lung metastases and support exploration of combinatorial therapies using inhaled rhIL-15.

Virtual medical student education and recruitment during the COVID-19 pandemic.

Due to the immediate need for social distancing, as well as widespread disruption in clinical practices, brought on by the novel severe acute respiratory syndrome coronavirus 2 (COVID-19) pandemic, medical student education rapidly shifted to a virtual format, which resulted in a variety of innovative and remotely accessible practices to address new restrictions on face-to-face education. Educators approached curriculum design seeking to replicate as much of the in-person experience as possible, and were faced with overcoming the challenges of replacing the innately hands-on nature of surgery with virtual operative and skills experiences. Restrictions on in-person visiting electives expedited the role of virtual education as a notable opportunity for medical student education and recruitment, with a variety of approaches to engaging undergraduate medical learners, including the use of live-streaming operative cases, virtual didactic curricula, and a rise in podcasts; web-based conferences; and virtual journal clubs. In addition to education, virtual outreach to medical students has become an essential tool in trainee recruitment and selection, and ongoing application of novel educational platforms will allow for new opportunities in multi-institutional collaboration and exchange with a multitude of benefits to future vascular surgery trainees. Our aim was to outline the resources and practices used to virtually teach and recruit medical students and the benefits of virtual rotations to the program and students.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase.

Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease. The protective role of PDI in Parkinson's Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

Trust in genomic data sharing among members of the general public in the UK, USA, Canada and Australia.

Trust may be important in shaping public attitudes to genetics and intentions to participate in genomics research and big data initiatives. As such, we examined trust in data sharing among the general public. A cross-sectional online survey collected responses from representative publics in the USA, Canada, UK and Australia (n = 8967). Participants were most likely to trust their medical doctor and less likely to trust other entities named. Company researchers were least likely to be trusted. Low, Variable and High Trust classes were defined using latent class analysis. Members of the High Trust class were more likely to be under 50 years, male, with children, hold religious beliefs, have personal experience of genetics and be from the USA. They were most likely to be willing to donate their genomic and health data for clinical and research uses. The Low Trust class were less reassured than other respondents by laws preventing exploitation of donated information. Variation in trust, its relation to areas of concern about the use of genomic data and potential of legislation are considered. These findings have relevance for efforts to expand genomic medicine and data sharing beyond those with personal experience of genetics or research participants.

Attitudes of publics who are unwilling to donate DNA data for research.

With the use of genetic technology, researchers have the potential to inform medical diagnoses and treatment in actionable ways. Accurate variant interpretation is a necessary condition for the utility of genetic technology to unfold. This relies on the ability to access large genomic datasets so that comparisons can be made between variants of interest. This can only be successful if DNA and medical data are donated by large numbers of people to 'research', including clinical, non-profit and for-profit research initiatives, in order to be accessed by scientists and clinicians worldwide. The objective of the 'Your DNA, Your Say' global survey is to explore public attitudes, values and opinions towards willingness to donate and concerns regarding the donation of one's personal data for use by others. Using a representative sample of 8967 English-speaking publics from the UK, the USA, Canada and Australia, we explore the characteristics of people who are unwilling (n = 1426) to donate their DNA and medical information, together with an exploration of their reasons. Understanding this perspective is important for making sense of the interaction between science and society. It also helps to focus engagement initiatives on the issues of concern to some publics.

'Your DNA, Your Say': global survey gathering attitudes toward genomics: design, delivery and methods.

Our international study, 'Your DNA, Your Say', uses film and an online cross-sectional survey to gather public attitudes toward the donation, access and sharing of DNA information. We describe the methodological approach used to create an engaging and bespoke survey, suitable for translation into many different languages. We address some of the particular challenges in designing a survey on the subject of genomics. In order to understand the significance of a genomic result, researchers and clinicians alike use external databases containing DNA and medical information from thousands of people. We ask how publics would like their 'anonymous' data to be used (or not to be used) and whether they are concerned by the potential risks of reidentification; the results will be used to inform policy.