Study of Alteplase for Respiratory Failure in SARS-CoV-2 COVID-19: A Vanguard Multicenter, Rapidly Adaptive, Pragmatic, Randomized Controlled Trial.

BACKGROUND: Pulmonary vascular microthrombi are a proposed mechanism of COVID-19 respiratory failure. We hypothesized that early administration of tissue plasminogen activator (tPA) followed by therapeutic heparin would improve pulmonary function in these patients. RESEARCH QUESTION: Does tPA improve pulmonary function in severe COVID-19 respiratory failure, and is it safe? STUDY DESIGN AND METHODS: Adults with COVID-19-induced respiratory failure were randomized from May14, 2020 through March 3, 2021, in two phases. Phase 1 (n = 36) comprised a control group (standard-of-care treatment) vs a tPA bolus (50-mg tPA IV bolus followed by 7 days of heparin; goal activated partial thromboplastin time [aPTT], 60-80 s) group. Phase 2 (n = 14) comprised a control group vs a tPA drip (50-mg tPA IV bolus, followed by tPA drip 2 mg/h plus heparin 500 units/h over 24 h, then heparin to maintain aPTT of 60-80 s for 7 days) group. Patients were excluded from enrollment if they had not undergone a neurologic examination or cross-sectional brain imaging within the previous 4.5 h to rule out stroke and potential for hemorrhagic conversion. The primary outcome was Pao2 to Fio2 ratio improvement from baseline at 48 h after randomization. Secondary outcomes included Pao2 to Fio2 ratio improvement of > 50% or Pao2 to Fio2 ratio of ≥ 200 at 48 h (composite outcome), ventilator-free days (VFD), and mortality. RESULTS: Fifty patients were randomized: 17 in the control group and 19 in the tPA bolus group in phase 1 and eight in the control group and six in the tPA drip group in phase 2. No severe bleeding events occurred. In the tPA bolus group, the Pao2 to Fio2 ratio values were significantly (P < .017) higher than baseline at 6 through 168 h after randomization; the control group showed no significant improvements. Among patients receiving a tPA bolus, the percent change of Pao2 to Fio2 ratio at 48 h (16.9% control [interquartile range (IQR), -8.3% to 36.8%] vs 29.8% tPA bolus [IQR, 4.5%-88.7%]; P = .11), the composite outcome (11.8% vs 47.4%; P = .03), VFD (0.0 [IQR, 0.0-9.0] vs 12.0 [IQR, 0.0-19.0]; P = .11), and in-hospital mortality (41.2% vs 21.1%; P = .19) did not reach statistically significant differences when compared with those of control participants. The patients who received a tPA drip did not experience benefit. INTERPRETATION: The combination of tPA bolus plus heparin is safe in severe COVID-19 respiratory failure. A phase 3 study is warranted given the improvements in oxygenation and promising observations in VFD and mortality. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT04357730; URL: www. CLINICALTRIALS: gov.

Langerhans cells serve as immunoregulatory cells by activating NKT cells.

Ultraviolet exposure alters the morphology and function of epidermal Langerhans cells (LCs), which play a role in UV-induced immune suppression. It is generally believed that UV exposure triggers the migration of immature LCs from the skin to the draining lymph nodes (LNs), where they induce tolerance. However, because most of the previous studies employed in vitro UV-irradiated LCs, the data generated may not adequately reflect what is happening in vivo. In this study, we isolated migrating LCs from the LNs of UV-irradiated mice and studied their function. We found prolonged LC survival in the LNs of UV-irradiated mice. LCs were necessary for UV-induced immune suppression because no immune suppression was observed in LC-deficient mice. Transferring LCs from UV-irradiated mice into normal recipient animals transferred immune suppression and induced tolerance. We found that LCs colocalized with LN NKT cells. No immune suppression was observed when LCs were transferred from UV-irradiated mice into NKT cell-deficient mice. NKT cells isolated from the LNs of UV-irradiated mice secreted significantly more IL-4 than NKT cells isolated from nonirradiated controls. Injecting the wild-type mice with anti-IL-4 blocked the induction of immune suppression. Our findings indicate that UV exposure activates the migration of mature LC to the skin draining LNs, where they induce immune regulation in vivo by activating NKT cells.

Agents that reverse UV-Induced immune suppression and photocarcinogenesis affect DNA repair.

UV exposure induces skin cancer, in part, by inducing immune suppression. Repairing DNA damage, neutralizing the activity of cis-urocanic acid, and reversing oxidative stress abrogate UV-induced immune suppression and skin cancer induction, suggesting that DNA, UCA, and lipid photo-oxidation serve as UV photoreceptors. What is not clear is whether signaling through each of these different photoreceptors activates independent pathways to induce biological effects or whether there is a common checkpoint where these pathways converge. Here, we show that agents known to reverse photocarcinogenesis and photoimmune suppression, such as platelet-activating factor (PAF) and serotonin (5-HT) receptor antagonists, regulate DNA repair. Pyrimidine dimer repair was accelerated in UV-irradiated mice injected with PAF and 5-HT receptor antagonists. Nucleotide excision repair (NER), as measured by unscheduled DNA synthesis, was accelerated by PAF and 5-HT receptor antagonists. Injecting PAF and 5-HT receptor antagonists into UV-irradiated Xeroderma pigmentosum complementation group A-deficient mice, which lack the enzymes responsible for NER, did not accelerate photoproduct repair. Similarly, UV-induced formation of 8-oxo-deoxyguanosine was reduced by PAF and 5-HT receptor antagonists. We conclude that PAF and 5-HT receptor antagonists accelerate DNA repair caused by UV radiation, which prevents immune suppression and interferes with photocarcinogenesis.

AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+ transporter.

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na(+), K(+), and Mn(2+) transport. We also report high-capacity uptake of (86)Rb(+) in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of (86)Rb(+) uptake in AtCCX3 cells by excess Na(+), K(+), and Mn(2+). Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl(2). Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na(+) accumulation and increased (86)Rb(+) transport. Uptake of (86)Rb(+) increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H(+)-dependent K(+) transporter with apparent Na(+) and Mn(2+) transport properties distinct from those of previously characterized plant transporters.

Inhibition of photocarcinogenesis by platelet-activating factor or serotonin receptor antagonists.

The UV radiation in sunlight is the primary cause of nonmelanoma skin cancer. Moreover, UV exposure induces immune suppression. Early steps in the cascade of events leading to immune suppression are the binding of UV-induced platelet-activating factor (PAF) to its receptor and the binding of cis-urocanic acid, a photoreceptor for UVB radiation, to the serotonin (5-HT(2A)) receptor. Here, we tested the hypothesis that blocking the binding of PAF and 5-HT(2A) to their receptors would also block skin cancer induction. Hairless mice were injected with PAF or serotonin receptor antagonists and then exposed to solar-simulated UV radiation. We noted a significant and substantial decrease in skin cancer incidence in mice treated with the PAF or 5-HT(2A) receptor antagonists. Also, the PAF and/or serotonin receptor antagonists blocked skin cancer progression. The PAF and serotonin receptor antagonists worked in a synergistic fashion to block skin cancer induction. We also measured the effect that injecting PAF and 5-HT(2A) receptor antagonists had on UV-induced skin damage after a single UV exposure. We noted a significant decrease in UV-induced hypertrophy, sunburn cell formation, and apoptosis when the mice were injected with PAF and/or 5-HT(2A) receptor antagonists. These data indicate that treating UV-irradiated mice with PAF and 5-HT(2A) receptor antagonists blocks skin cancer induction in vivo, in part by reversing UV-induced damage to the skin and by preventing the induction of immune suppression.

Dermal dendritic cells, and not Langerhans cells, play an essential role in inducing an immune response.

Langerhans cells (LCs) serve as epidermal sentinels of the adaptive immune system. Conventional wisdom suggests that LCs encounter Ag in the skin and then migrate to the draining lymph nodes, where the Ag is presented to T cells, thus initiating an immune response. Platelet-activating factor (PAF) is a phospholipid mediator with potent biological effects. During inflammation, PAF mediates recruitment of leukocytes to inflammatory sites. We herein tested a hypothesis that PAF induces LC migration. Applying 2,4-dinitro-1-fluorobenzene (DNFB) to wild-type mice activated LC migration. In contrast, applying DNFB to PAF receptor-deficient mice or mice injected with PAF receptor antagonists failed to induce LC migration. Moreover, after FITC application the appearance of hapten-laden LCs (FITC+, CD11c+, Langerin+) in the lymph nodes of PAF receptor-deficient mice was significantly depressed compared with that found in wild-type mice. LC chimerism indicates that the PAF receptor on keratinocytes but not LCs is responsible for LC migration. Contrary to the diminution of LC migration in PAF receptor-deficient mice, we did not observe any difference in the migration of hapten-laden dermal dendritic cells (FITC+, CD11c+, Langerin-) into the lymph nodes of PAF receptor-deficient mice. Additionally, the contact hypersensitivity response generated in wild-type or PAF receptor-deficient mice was identical. Finally, dermal dendritic cells, but not LCs isolated from the draining lymph nodes after hapten application, activated T cell proliferation. These findings suggest that LC migration may not be responsible for the generation of contact hypersensitivity and that dermal dendritic cells may play a more important role.

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3.

Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. Structure/function analysis of the Arabidopsis H+/cation exchanger, CAX1, revealed that a nine amino acid region (87-95) is involved in CAX1-mediated Ca2+ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca2+ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca2+ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca2+ transport capability compared to CAX3-I. The entire Cad of CAX3 (87-95) inserted into CAX1 abolishes CAX1-mediated Ca2+ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca2+ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca2+ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.

Distinct N-terminal regulatory domains of Ca(2+)/H(+) antiporters.

The regulation of intracellular Ca(2+) levels is achieved in part by high-capacity vacuolar Ca(2+)/H(+) antiporters. An N-terminal regulatory region (NRR) on the Arabidopsis Ca(2+)/H(+) antiporter CAX1 (cation exchanger 1) has been shown previously to regulate Ca(2+) transport by a mechanism of N-terminal auto-inhibition. Here, we examine the regulation of other CAX transporters, both within Arabidopsis and from another plant, mung bean (Vigna radiata), to ascertain if this mechanism is commonly used among Ca(2+)/H(+) antiporters. Biochemical analysis of mung bean VCAX1 expressed in yeast (Saccharomyces cerevisiae) showed that N-terminal truncated VCAX1 had approximately 70% greater antiport activity compared with full-length VCAX1. A synthetic peptide corresponding to the NRR of CAX1, which can strongly inhibit Ca(2+) transport by CAX1, could not dramatically inhibit Ca(2+) transport by truncated VCAX1. The N terminus of Arabidopsis CAX3 was also shown to contain an NRR. Additions of either the CAX3 or VCAX1 regulatory regions to the N terminus of an N-terminal truncated CAX1 failed to inhibit CAX1 activity. When fused to N-terminal truncated CAX1, both the CAX3 and VCAX1 regulatory regions could only auto-inhibit CAX1 after mutagenesis of specific amino acids within this NRR region. These findings demonstrate that N-terminal regulation is present in other plant CAX transporters, and suggest distinct regulatory features among these transporters.