Lymphatic coagulation and neutrophil extracellular traps in lung-draining lymph nodes of COVID-19 decedents.

Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.

A Spatiotemporal Analytical Outlook of the Exposure to Air Pollution and COVID-19 Mortality in the USA.

The world is experiencing a pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), also known as COVID-19. The USA is also suffering from a catastrophic death toll from COVID-19. Several studies are providing preliminary evidence that short- and long-term exposure to air pollution might increase the severity of COVID-19 outcomes, including a higher risk of death. In this study, we develop a spatiotemporal model to estimate the association between exposure to fine particulate matter PM2.5 and mortality accounting for several social and environmental factors. More specifically, we implement a Bayesian zero-inflated negative binomial regression model with random effects that vary in time and space. Our goal is to estimate the association between air pollution and mortality accounting for the spatiotemporal variability that remained unexplained by the measured confounders. We applied our model to four regions of the USA with weekly data available for each county within each region. We analyze the data separately for each region because each region shows a different disease spread pattern. We found a positive association between long-term exposure to PM2.5 and the mortality from the COVID-19 disease for all four regions with three of four being statistically significant. Data and code are available at our GitHub repository. Supplementary materials accompanying this paper appear on-line. Supplementary Information: The online version contains supplementary material available at 10.1007/s13253-022-00487-1.

Effect of Human Synovial Fluid From Osteoarthritis Patients and Healthy Individuals on Lymphatic Contractile Activity.

The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining overall joint homeostasis; however, there is some evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from osteoarthritis patients (OASF). Here, we utilized OASF and SF derived from healthy (non-OA) individuals (healthy SF (HSF)) to investigate potential effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractile activity of rat femoral LVs (RFLVs) ex vivo. Dilutions of both OASF and HSF containing serum resulted in a similar LEC migratory response to the physiologically endothelial basal medium-treated LECs (endothelial basal medium containing serum) in vitro. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 h of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractile activity (both tonic and phasic contractions). The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Public Awareness of and Support for the Use of Wastewater for SARS-CoV-2 Monitoring: A Community Survey in Louisville, Kentucky.

The majority of sewer systems in the United States and other countries are operated by public utilities. In the absence of any regulation, the public perception of wastewater monitoring for population health biomarkers is an important consideration for a public utility commission when allocating resources for this purpose. We conducted a survey in August 2021 as part of an ongoing COVID-19 community prevalence study in Louisville/Jefferson County, KY, US. The survey comprised seven questions about wastewater awareness and privacy concerns and was sent to approximately 35 000 households randomly distributed within the county. A total of 1220 adults were involved in the probability sample, and data from 981 respondents were used in the analysis. A total of 2444 adults additionally responded to the convenience sample, and data from 1751 respondents were used in the analysis. The samples were weighted to obtain estimates representative of all adults in the county. Public awareness of tracking the virus that causes COVID-19 in sewers was low. Opinions strongly support the public disclosure of monitoring results. Responses showed that people more strongly supported measurements in the largest areas (>50 000 households), typically representing population levels found in a large community wastewater treatment plant. Those with a history of COVID-19 infection were more likely to support highly localized monitoring. Understanding wastewater surveillance strategies and privacy concern thresholds requires an in-depth and comprehensive analysis of public opinion for continued success and effective public health monitoring.

Lymphatic injury alters the contractility and mechanosensitivity of collecting lymphatics to intermittent pneumatic compression.

KEY POINTS: We present the first in vivo evidence that lymphatic contraction can entrain with an external oscillatory mechanical stimulus. Lymphatic injury can alter collecting lymphatic contractility, but not much is known about how its mechanosensitivity to external pressure is affected, which is crucial given the current pressure application methods for treating lymphoedema. We show that oscillatory pressure waves (OPW), akin to intermittent pneumatic compression (IPC) therapy, optimally entrain lymphatic contractility and modulate function depending on the frequency and propagation speed of the OPW. We show that the OPW-induced entrainment and contractile function in the intact collecting lymphatics are enhanced 28 days after a contralateral lymphatic ligation surgery. The results show that IPC efficacy can be improved through proper selection of OPW parameters, and that collecting lymphatics adapt their function and mechanosensitivity after a contralateral injury, switching their behaviour to a pump-like configuration that may be more suited to the altered microenvironment. ABSTRACT: Intermittent pneumatic compression (IPC) is commonly used to control the swelling due to lymphoedema, possibly modulating the collecting lymphatic function. Lymphoedema causes lymphatic contractile dysfunction, but the consequent alterations in the mechanosensitivity of lymphatics to IPC is not known. In the present work, the spatiotemporally varying oscillatory pressure waves (OPW) generated during IPC were simulated to study the modulation of lymphatic function by OPW under physiological and pathological conditions. OPW with three temporal frequencies and three propagation speeds were applied to rat tail collecting lymphatics. The entrainment of the lymphatics to OPW was significantly higher at a frequency of 0.05 Hz compared with 0.1 Hz and 0.2 Hz (P = 0.0054 and P = 0.014, respectively), but did not depend on the OPW propagation speed. Lymphatic function was significantly higher at a frequency of 0.05 Hz and propagation speed of 2.55 mm/s (P = 0.015). Exogenous nitric oxide was not found to alter OPW-induced entrainment. A contralateral lymphatic ligation surgery was performed to simulate partial lymphatic injury in rat tails. The intact vessels showed a significant increase in entrainment to OPW, 28 days after ligation (compared with sham) (P = 0.016), with a similar increase in lymphatic transport function (P = 0.0029). The results suggest an enhanced mechanosensitivity of the lymphatics, along with a transition to a pump-like behaviour, in response to a lymphatic injury. These results enhance our fundamental understanding of how lymphatic mechanosensitivity assists the coordination of lymphatic contractility and how this might be leveraged in IPC therapy.

A Practical Overview and Reporting Strategies for Statistical Analysis of Survival Studies.

Survival (time-to-event) analysis is commonly used in clinical research. Key features of performing a survival analysis include checking proportional hazards assumptions, reporting CIs for hazards ratios and relative risks, graphically displaying the findings, and analyzing with consideration of competing risks. This article provides a brief overview of important statistical considerations for survival analysis. Censoring schemes, different methods of survival function estimation, and ways to compare survival curves are described. We also explain competing risk and how to model survival data in the presence of it. Different kinds of bias that influence survival estimation and avenues to model the data under these circumstances are also described. Several analysis techniques are accompanied by graphical representations illustrating proper reporting strategies. We provide a list of guiding statements for researchers and reviewers.

A Practical Overview of Case-Control Studies in Clinical Practice.

Case-control studies are one of the major observational study designs for performing clinical research. The advantages of these study designs over other study designs are that they are relatively quick to perform, economical, and easy to design and implement. Case-control studies are particularly appropriate for studying disease outbreaks, rare diseases, or outcomes of interest. This article describes several types of case-control designs, with simple graphical displays to help understand their differences. Study design considerations are reviewed, including sample size, power, and measures associated with risk factors for clinical outcomes. Finally, we discuss the advantages and disadvantages of case-control studies and provide a checklist for authors and a framework of considerations to guide reviewers' comments.

Functional modeling of pedaling kinematics for the Stroke patients.

Understanding deficits in motor control through the analysis of pedaling biomechanics plays a key role in the treatment of stroke patients. A thorough study of the impact of different exercise patterns and workloads on the change between pre- and post-treatment movement patterns in the patients is therefore of utmost importance to the clinicians. The objective of this study was to analyze the difference between pre- and post-treatment pedaling torques when the patients are subject to different exercise groups with varying workloads. The effects of affected vs unaffected side along with the covariates age and BMI have also been accounted for in this work. Two different three-way ANOVA-based approaches have been implemented here. In the first approach, a random projection-based ANOVA technique has been performed treating the pedaling torques as functional response, whereas the second approach utilizes distance measures to summarize the difference between pre- and post-treatment torques and perform nonparametric tests on it. Bayesian bootstrap has been used here to perform tests on the median distance. A group of stroke patients have been studied in the Cleveland Clinic categorizing them into different exercise groups and workload patterns. The data obtained have been analyzed with the aforementioned techniques, and the results have been reported here. These techniques turn out to be promising and will help clinicians recommend personalized treatment to stroke patients for optimal results.

Entrainment of Lymphatic Contraction to Oscillatory Flow.

Lymphedema, a disfiguring condition characterized by an asymmetrical swelling of the limbs, is suspected to be caused by dysfunctions in the lymphatic system. A possible source of lymphatic dysfunction is the reduced mechanosensitivity of lymphangions, the spontaneously contracting units of the lymphatic system. In this study, the entrainment of lymphangions to an oscillatory wall shear stress (OWSS) is characterized in rat thoracic ducts in relation to their shear sensitivity. The critical shear stress above which the thoracic ducts show a substantial inhibition of contraction was found to be significantly negatively correlated to the diameter of the lymphangion. The entrainment of the lymphangion to an applied OWSS was found to be significantly dependent on the difference between the applied frequency and the intrinsic frequency of contraction of the lymphangion. The strength of the entrainment was also positively correlated to the applied shear stress when the applied shear was less than the critical shear stress of the vessel. The ejection fraction and fractional pump flow were also affected by the difference between the frequency of the applied OWSS and the vessel's intrinsic contraction frequency. The results suggest an adaptation of the lymphangion contractility to the existing oscillatory shear stress as a function of its intrinsic contractility and shear sensitivity. These adaptations might be crucial to ensure synchronized contraction of lymphangions through mechanosensitive means and might help explain the lymphatic dysfunctions that result from impaired mechanosensitivity.

Mechanotransduction activates canonical Wnt/ß-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves.

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/ß-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of ß-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/ß-catenin signaling in LECs. In turn, Wnt/ß-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking ß-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/ß-catenin signaling and, in turn, FOXC2.

Effects of dynamic shear and transmural pressure on wall shear stress sensitivity in collecting lymphatic vessels.

Given the known mechanosensitivity of the lymphatic vasculature, we sought to investigate the effects of dynamic wall shear stress (WSS) on collecting lymphatic vessels while controlling for transmural pressure. Using a previously developed ex vivo lymphatic perfusion system (ELPS) capable of independently controlling both transaxial pressure gradient and average transmural pressure on an isolated lymphatic vessel, we imposed a multitude of flow conditions on rat thoracic ducts, while controlling for transmural pressure and measuring diameter changes. By gradually increasing the imposed flow through a vessel, we determined the WSS at which the vessel first shows sign of contraction inhibition, defining this point as the shear stress sensitivity of the vessel. The shear stress threshold that triggered a contractile response was significantly greater at a transmural pressure of 5 cmH2O (0.97 dyne/cm(2)) than at 3 cmH2O (0.64 dyne/cm(2)). While contraction frequency was reduced when a steady WSS was applied, this inhibition was reversed when the applied WSS oscillated, even though the mean wall shear stresses between the conditions were not significantly different. When the applied oscillatory WSS was large enough, flow itself synchronized the lymphatic contractions to the exact frequency of the applied waveform. Both transmural pressure and the rate of change of WSS have significant impacts on the contractile response of lymphatic vessels to flow. Specifically, time-varying shear stress can alter the inhibition of phasic contraction frequency and even coordinate contractions, providing evidence that dynamic shear could play an important role in the contractile function of collecting lymphatic vessels.