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"Freeman, W. David"
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Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes
Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.
Journal Article
Extracellular vesicles in diabetes mellitus induce alterations in endothelial cell morphology and migration
Background
Inflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. Extracellular vesicles (EVs) are nano-sized particles that contain molecular cargo and circulate in the blood. Here, we examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells.
Methods
We quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort.
Results
We found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and β-cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals.
Conclusions
Higher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease.
Journal Article
The eSAH score: a simple practical predictive model for SAH mortality and outcomes
We developed a simple quantifiable scoring system that predicts aneurysmal subarachnoid hemorrhage (aSAH) mortality, delayed cerebral ischemia (DCI), and modified Rankin scale (mRS) outcomes using readily available SAH admission data with SAH volume (SAHV) measured on computed tomography (CT). We retrospectively analyzed a cohort of 277 patients with aSAH admitted at our Comprehensive Stroke Center at Mayo Clinic in Jacksonville, Florida, between January 5, 2012, and February 24, 2022. We developed a mathematical radiographic model SAHV that measures basal cisternal SAH blood volume using a derivation of the ABC/2 ellipsoid formula (A = width/thickness, B = length, C = vertical extension) on noncontrast CT, which we previously demonstrated is comparable to pixel-based manual segmentation on noncontrast CT. Data were analyzed using
t
test, χ
2
test, receiver operator characteristics curve, and area under the curve (AUC) analysis. Multivariate logistic regression analysis with stepwise elimination of variables not contributing to the model (0.05 significance level for entry into the model) was used to develop an enhanced SAH (eSAH) scoring system. Using multivariate logistic regression, we found that age, Glasgow Coma Scale score, and SAHV were significantly associated with mRS outcomes at discharge, in-hospital DCI, and in-hospital mortality. Using these factors, we developed a weighted eSAH score, ranging from 0 to 5, that was strongly predictive of mRS outcomes (AUC = 0.89), DCI (AUC = 0.75), and in-hospital mortality (AUC = 0.88). Our proposed eSAH score, a simple quantitative model based on SAHV, Glasgow Coma Scale score, and age, appears to predict mortality and outcomes in patients with aSAH. A larger cohort validation study is planned.
Journal Article
Association between GDF15, poverty and mortality in urban middle-aged African American and white adults
Mortality disparities are influenced by race and poverty. There is limited information about whether poverty influences biologic markers of mortality risk. Emerging data suggests that growth differentiation factor 15 (GDF15) is associated with mortality; however, the interplay between GDF15, sociodemographic factors and mortality is not known. We sought to evaluate the interactions between GDF15 and sex, race and poverty status on mortality. Serum GDF15 was measured in 1036 African American and white middle-aged men and women above and below 125% of the Federal poverty status from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Multivariable adjusted Cox regression models were used to assess the association between log-transformed GDF15 (logGDF15) and 12-year mortality outcomes (all-cause, cardiovascular- and cancer-specific outcomes) and interactions with sex, race and poverty status. Likelihood ratio tests were used to assess significance of the interaction terms. Median GDF15 was 655.2 pg/mL (IQR = 575.1). During 12.2 years of follow-up, 331 died of which 94 cardiovascular- and 87 were cancer-specific deaths. One unit of increase in logGDF15 was associated with a hazard ratio for all-cause mortality, cardiovascular- and cancer-specific mortality of 2.26 (95% confidence interval [CI], 1.94-2.64), 2.74 (95%CI, 2.06-3.63) and 1.41 (95%CI, 1.00-2.00), respectively. There was an interaction between logGDF15 and poverty status on all-cause mortality (p<0.05). The GDF15xpoverty status interaction term improved model calibration for all-cause mortality. Our study provides the first evidence that the effect of elevated GDF15 on all-cause mortality is modified by poverty status.
Journal Article
Safety, feasibility, and impact on the gut microbiome of kefir administration in critically ill adults
Background
Dysbiosis of the gut microbiome is frequent in the intensive care unit (ICU), potentially leading to a heightened risk of nosocomial infections. Enhancing the gut microbiome has been proposed as a strategic approach to mitigate potential adverse outcomes. While prior research on select probiotic supplements has not successfully shown to improve gut microbial diversity, fermented foods offer a promising alternative. In this open-label phase I safety and feasibility study, we examined the safety and feasibility of kefir as an initial step towards utilizing fermented foods to mitigate gut dysbiosis in critically ill patients.
Methods
We administered kefir in escalating doses (60 mL, followed by 120 mL after 12 h, then 240 mL daily) to 54 critically ill patients with an intact gastrointestinal tract. To evaluate kefir’s safety, we monitored for gastrointestinal symptoms. Feasibility was determined by whether patients received a minimum of 75% of their assigned kefir doses. To assess changes in the gut microbiome composition following kefir administration, we collected two stool samples from 13 patients: one within 72 h of admission to the ICU and another at least 72 h after the first stool sample.
Results
After administering kefir, none of the 54 critically ill patients exhibited signs of kefir-related bacteremia. No side effects like bloating, vomiting, or aspiration were noted, except for diarrhea in two patients concurrently on laxatives. Out of the 393 kefir doses prescribed for all participants, 359 (91%) were successfully administered. We were able to collect an initial stool sample from 29 (54%) patients and a follow-up sample from 13 (24%) patients. Analysis of the 26 paired samples revealed no increase in gut microbial α-diversity between the two timepoints. However, there was a significant improvement in the Gut Microbiome Wellness Index (GMWI) by the second timepoint (
P
= 0.034, one-sided Wilcoxon signed-rank test); this finding supports our hypothesis that kefir administration can improve gut health in critically ill patients. Additionally, the known microbial species in kefir were found to exhibit varying levels of engraftment in patients’ guts.
Conclusions
Providing kefir to critically ill individuals is safe and feasible. Our findings warrant a larger evaluation of kefir’s safety, tolerability, and impact on gut microbiome dysbiosis in patients admitted to the ICU.
Trial registration
NCT05416814; trial registered on June 13, 2022.
Journal Article
Refractory and Super-Refractory Status Epilepticus—an Update
Status epilepticus is a medical emergency with a high mortality. Early recognition and initiation of treatment leads to a better response and may improve outcomes. Refractory status epilepticus is defined as recurrent seizure activity despite two appropriately selected and dosed antiepileptic drugs including a benzodiazepine. The term “super-refractory status epilepticus” was introduced during the London–Innsbruck Colloquium on status epilepticus in 2011 and refers to status epilepticus that continues or recurs 24 h or more after the initiation of treatment with anesthetic antiepileptic drugs. This includes cases in which seizure control is attained after induction of anesthesia but recurs on weaning the patient off the anesthetic agent. This article reviews the approach to refractory status epilepticus and super-refractory status epilepticus, including management as well as common pathophysiological causes of these entities.
Journal Article
CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress
Background
CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4
L75A
-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells.
Methods
We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4
L75A
-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4
L75A
-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4
L75A
-Fc, which represents a candidate therapeutic approach.
Results
ALK4
L75A
-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4
L75A
-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden.
Conclusions
Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4
L75A
-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.
Journal Article
Early Progressive Mobilization of Patients with External Ventricular Drains: Safety and Feasibility
Background/Objective
Early mobilization of critically ill patients has been shown to improve functional outcomes. Neurosurgery patients with an external ventricular drain (EVD) due to increased intracranial pressure often remain on bed rest while EVD remains in place. The prevalence of mobilizing patients with EVD has not been described, and the literature regarding the safety and feasibility of mobilizing patients with EVDs is limited. The aim of our study was to describe the outcomes and adverse events of the first mobilization attempt in neurosurgery patients with EVD who participated in early functional mobilization with physical therapy or occupational therapy.
Methods
We performed a single-site, retrospective chart review of 153 patients who underwent placement of an EVD. Hemodynamically stable patients deemed appropriate for mobilization by physical or occupational therapy were included. Mobilization and activity details were recorded.
Results
The most common principal diagnoses were subarachnoid hemorrhage (61.4%) and intracerebral hemorrhage (17.0%) requiring EVD for symptomatic hydrocephalus. A total of 117 patients were mobilized (76.5%), and the median time to first mobilization after EVD placement in this group of 117 patients was 38 h. Decreased level of consciousness was the most common reason for lack of mobilization. The highest level of mobility on the patient’s first attempt was ambulation (43.6%), followed by sitting on the side of the bed (30.8%), transferring to a bedside chair (17.1%), and standing up from the side of the bed (8.5%). No major safety events, such as EVD dislodgment, occurred in any patient. Transient adverse events with mobilization were infrequent at 6.9% and had no permanent neurological sequelae and were mostly headache, nausea, and transient diastolic blood pressure elevation.
Conclusion
Early progressive mobilization of neurosurgical intensive care unit patients with external ventricular drains appears safe and feasible.
Journal Article
Mcam stabilizes a luminal progenitor-like breast cancer cell state via Ck2 control and Src/Akt/Stat3 attenuation
Cell state control is crucial for normal tissue development and cancer cell mimicry of stem/progenitor states, contributing to tumor heterogeneity, therapy resistance, and progression. Here, we demonstrate that the cell surface glycoprotein Mcam maintains the tumorigenic luminal progenitor (LP)-like epithelial cell state, leading to Basal-like mammary cancers. In the Py230 mouse mammary carcinoma model, Mcam knockdown (KD) destabilized the LP state by deregulating the Ck2/Stat3 axis, causing a switch to alveolar and basal states, loss of an estrogen-sensing subpopulation, and resistance to tamoxifen—an effect reversed by Ck2 and Stat3 inhibitors. In vivo, Mcam KD blocked generation of Basal-like tumors and Sox10+Krt14+ cells. In human tumors, MCAM loss was largely exclusive of the Basal-like subtype, linked instead to proliferative Luminal subtypes, including often endocrine-resistant Luminal B cancers. This study has implications for developing therapies targeting MCAM, CK2, and STAT3 and their likely effective contexts.
Journal Article