Hydroxychloroquine is an antimalarial drug being tested as a potential treatment for the novel coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2. Although the efficacy of hydroxychloroquine for COVID-19 remains uncertain, it may serve as a potential prophylactic agent especially in those at high risk, such as healthcare workers, household contacts of infected patients, and the immunocompromised. Our aim was to identify possible hydroxychloroquine dosing regimens through simulation in those at high risk of infections by optimizing exposures above the in vitro generated half maximal effective concentration (EC 50 ) and to help guide researchers in dose-selection for COVID-19 prophylactic studies. To maintain weekly troughs above EC 50 in > 50% of subjects at steady-state in a pre-exposure prophylaxis setting, an 800 mg loading dose followed by 400 mg twice or 3 times weekly is required. In an exposure driven, postexposure prophylaxis setting, 800 mg loading dose followed in 6 hours by 600 mg, then 600 mg daily for 4 more days achieved daily troughs above EC 50 in > 50% subjects. These doses are higher than recommended for malaria chemoprophylaxis, and clinical trials are needed to establish safety and efficacy.

The lymphatic system maintains tissue homeostasis by transporting interstitial fluid, lipids and debris from tissues to the main circulation, and delivers antigen and antigen presenting cells to local lymph nodes where they elicit immune responses. During inflammation, lymph flow increases to limit edema and prevents tissue antigen-presenting cell transport. Lymphatics also adjust their contractile activity to increase fluid transfer during acute inflammation. Conversely, chronic inflammation can provoke lymphostasis, which might limit pathogen spread within the circulation; however, decreased lymph flow leads to the persistence of immune cells and mediators in tissues to intensifying injury. Here, we review lymphatic structure function within the gut, heart and central nervous system, discussing potential roles of these lymphatics in the etiology of inflammatory bowel disease, myocarditis and neurovascular disease, and as novel targets for therapeutic management of several disease states.

Theiler's murine encephalomyelitis virus (TMEV) induces different diseases in the central nervous system (CNS) and heart, depending on the mouse strains and time course, with cytokines playing key roles for viral clearance and immune-mediated pathology (immunopathology). In SJL/J mice, TMEV infection causes chronic TMEV-induced demyelinating disease (TMEV-IDD) in the spinal cord about 1 month post-inoculation (p.i.). Unlike other immunopathology models, both pro- and anti-inflammatory cytokines can play dual roles in TMEV-IDD. Pro-inflammatory cytokines play beneficial roles in viral clearance while they are also detrimental in immune-mediated demyelination. Anti-inflammatory cytokines suppress not only protective anti-viral immune responses but also detrimental autoreactive immune responses. Conversely, in C3H mice, TMEV infection induces a non-CNS disease, myocarditis, with three distinctive phases: phase I, viral pathology with interferon and chemokine responses; phase II, immunopathology mediated by acquired immune responses; and phase III, cardiac fibrosis. Although the exact mechanism(s) by which a single virus, TMEV, induces these different diseases in different organs is unclear, our bioinformatics approaches, especially principal component analysis (PCA) of transcriptome data, allow us to identify the key factors contributing to organ-specific immunopathology. The PCA demonstrated that in vitro infection of a cardiomyocyte cell line reproduced the transcriptome profile of phase I in TMEV-induced myocarditis; distinct interferon/chemokine-related responses were induced in vitro in TMEV-infected cardiomyocytes, but not in infected neuronal cells. In addition, the PCA of the in vivo CNS transcriptome data showed that decreased lymphatic marker expressions were weakly associated with inflammation in TMEV infection. Here, dysfunction of lymphatic vessels is shown to potentially contribute to immunopathology by delaying the clearance of cytokines and immune cells from the inflammatory site, although this can also confine the virus at these sites, preventing virus spread via lymphatic vessels. On the other hand, in the heart, dysfunction of lymphatics was associated with reduced lymphatic muscle contractility provoked by pro-inflammatory cytokines. Therefore, TMEV infection may induce different patterns of cytokine expressions as well as lymphatic vessel dysfunction by rather different mechanisms between the CNS and heart, which might explain observed patterns of organ-specific immunopathology.

Background and Purpose The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL‐1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC). Experimental Approach We measured IL‐1β in colon‐conditioned media (CM) from acute (AC‐CM, dextran sodium sulfate) and chronic (CC‐CM, T‐cell transfer) colitis‐induced mice and corresponding controls (Con‐AC/CC‐CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h‐IL‐1β or h‐TNF‐α (5, 10, 20 ng·mL−1), with or without COX inhibitors [TFAP (10−5 M), diclofenac (0.2 × 10−5 M)], PGE2 (10−5 M)], IL‐1‐receptor antagonist, Anakinra (5 μg·mL−1), or a selective prostanoid EP4 receptor antagonist, GW627368X (10−6 and 10−7 M). Key Results Tonic contractility of RMLMC was reduced by AC‐ and CC‐CM compared with corresponding control culture media, Con‐AC/CC‐CM. IL‐1β or TNF‐α was not found in Con‐AC/CC‐CM, but detected in AC‐ and CC‐CM. h‐IL‐1β concentration‐dependently decreased RMLMC contractility, whereas h‐TNF‐α showed no effect. Anakinra blocked h‐IL‐1β‐induced RMLMC relaxation, and with AC‐CM, restored contractility to RMLMC. IL‐1β increased COX‐2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h‐IL‐1β. Conversely, COX‐2 and EP4 receptor inhibition reversed relaxation induced by IL‐1β. Conclusions and Implications The IL‐1β‐induced decrease in RMLMC tonic contraction was COX‐2 dependent, and mediated by PGE2. In experimental colitis, IL‐1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC‐CM and pharmacological blockade of IL‐1β restored tonic contraction.

Aims The aim of this study was to characterize the pharmacokinetic/pharmacodynamic relationships of cortisol and the adrenal biomarkers 17‐hydroxyprogesterone and androstenedione in children with congenital adrenal hyperplasia (CAH). Methods A nonlinear mixed‐effect modelling approach was used to analyse cortisol, 17‐hydroxyprogesterone and androstenedione concentrations obtained over 6 hours from children with CAH (n = 50). A circadian rhythm was evident and the model leveraged literature information on circadian rhythm in untreated children with CAH. Indirect response models were applied in which cortisol inhibited the production rate of all three compounds using an Imax model. Results Cortisol was characterized by a one‐compartment model with apparent clearance and volume of distribution estimated at 22.9 L/h/70 kg and 41.1 L/70 kg, respectively. The IC50 values of cortisol concentrations for cortisol, 17‐hydroxyprogesterone and androstenedione were estimated to be 1.36, 0.45 and 0.75 μg/dL, respectively. The inhibitory effect was found to be more potent on 17OHP than D4A, and the IC50 values were higher in salt‐wasting subjects than simple virilizers. Production rates of cortisol, 17‐hydroxyprogesterone and androstenedione were higher in simple‐virilizer subjects. Half‐lives of cortisol, 17‐hydroxyprogesterone and androstenedione were 60, 47 and 77 minutes, respectively. Conclusion Rapidly changing biomarker responses to cortisol concentrations highlight that single measurements provide volatile information about a child's disease control. Our model closely captured observed cortisol, 17‐hydroxyprogesterone and androstenedione concentrations. It can be used to predict concentrations over 24 hours and allows many novel exposure metrics to be calculated, e.g., AUC, AUC‐above‐threshold, time‐within‐range, etc. Our long‐range goal is to uncover dose–exposure–outcome relationships that clinicians can use in adjusting hydrocortisone dose and timing.

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The role of lymphatic vessels in myocarditis is largely unknown, while it has been shown to play a key role in other inflammatory diseases. We aimed to investigate the role of lymphatic vessels in myocarditis using in vivo model induced with Theiler's murine encephalomyelitis virus (TMEV) and in vitro model with rat cardiac lymphatic muscle cells (RCLMC). In the TMEV model, we found that upregulation of a set of inflammatory mediator genes, including interleukin (IL)-1β, tumor necrosis factor (TNF)-αand COX-2 were associated with disease activity. Thus, using in vitro collagen gel contraction assays, we decided to clarify the role(s) of these mediators by testing contractility of RCLMC in response to IL-1β and TNF-α individually and in combination, in the presence or absence of: IL-1 receptor antagonist (Anakinra); cyclooxygenase (COX) inhibitors inhibitors (TFAP, diclofenac and DuP-697). IL-1β impaired RCLMC contractility dose-dependently, while co-incubation with both IL-1β and TNF-α exhibited synergistic effects in decreasing RCLMC contractility with increased COX-2 expression. Anakinra maintained RCLMC contractility; Anakinra blocked the mobilization of COX-2 induced by IL-1β with or without TNF-α. COX-2 inhibition blocked the IL-1β-mediated decrease in RCLMC contractility. Mechanistically, we found that IL-1β increased prostaglandin (PG) E release dose-dependently, while Anakinra blocked IL-1β mediated PGE release. Using prostaglandin E receptor 4 (EP4) receptor antagonist, we demonstrated that EP4 receptor blockade maintained RCLMC contractility following IL-1β exposure. Our results indicate that IL-1β reduces RCLMC contractility via COX-2/PGE signaling with synergistic cooperation by TNF-α. These pathways may help provoke inflammatory mediator accumulation within the heart, driving progression from acute myocarditis into dilated cardiomyopathy.

Placental vascular dysfunction has been linked to insufficiency/deficiency of maternal vitamin D levels during pregnancy. In contrast, sufficient maternal vitamin D levels have shown beneficial effects on pregnancy outcomes. To study the role of vitamin D in pregnancy, we tested our hypothesis that vitamin D exerts beneficial effects on placental vasculature. We examined expression of CYP2R1, CYP27B1, vitamin D receptor (VDR), and CYP24A1 in placental vascular smooth muscle cells (VSMCs) in response to 1,25(OH)2D3. We found that VDR expression was inducible, CYP27B1 expression was dose-dependently down-regulated, and CYP24A1 expression was dose-dependently up-regulated in cells treated with 1,25(OH)2D3. These data suggest a feedback autoregulatory system of vitamin D existing in placental VSMCs. Using a VSMC/collagen-gel contraction assay, we evaluated the effect of 1,25(OH)2D3 on placental VSMC contractility. We found that, similar to losartan, 1,25(OH)2D3 could diminish angiotensin II-induced cell contractility. The mechanism of 1,25(OH)2D3-mediated VSMC relaxation was further explored by examination of Rho-associated protein kinase 1 (ROCK1)/phosphorylation of myosin phosphatase target subunit 1 (MYPT1) pathway molecules. Our results showed that p-MYPT1Thr853 and p-MYPT1Thr696 were undetectable. However, p-MYPT1Ser507, but not p-MYPT1Ser668, was significantly up-regulated in cells treated with losartan plus angiotensin II. Similar effects were also seen in cells treated with 1,25(OH)2D3 plus angiotensin II or 1,25(OH)2D3 plus losartan plus angiotensin II. Because MYPT1 serine phosphorylation could activate myosin light chain phosphatase (MLCP), and MLCP activation is an important regulatory machinery of smooth muscle cell relaxation, up-regulation of MYPT1Ser507 phosphorylation could be a mechanism of vitamin D and/or losartan mediated placental VSMC relaxation.