Erythropoietin as candidate for supportive treatment of severe COVID-19.

In light of the present therapeutic situation in COVID-19, any measure to improve course and outcome of seriously affected individuals is of utmost importance. We recap here evidence that supports the use of human recombinant erythropoietin (EPO) for ameliorating course and outcome of seriously ill COVID-19 patients. This brief expert review grounds on available subject-relevant literature searched until May 14, 2020, including Medline, Google Scholar, and preprint servers. We delineate in brief sections, each introduced by a summary of respective COVID-19 references, how EPO may target a number of the gravest sequelae of these patients. EPO is expected to: (1) improve respiration at several levels including lung, brainstem, spinal cord and respiratory muscles; (2) counteract overshooting inflammation caused by cytokine storm/ inflammasome; (3) act neuroprotective and neuroregenerative in brain and peripheral nervous system. Based on this accumulating experimental and clinical evidence, we finally provide the research design for a double-blind placebo-controlled randomized clinical trial including severely affected patients, which is planned to start shortly.

Effects of non-fatiguing respiratory muscle loading induced by expiratory flow limitation during strenuous incremental cycle exercise on metabolic stress and circulating natural killer cells.

Exercise induces release of cytokines and increase of circulating natural killers (NK) lymphocyte during strong activation of respiratory muscles. We hypothesised that non-fatiguing respiratory muscle loading during exercise causes an increase in NK cells and in metabolic stress indices. Heart rate (HR), ventilation (VE), oesophageal pressure (Pes), oxygen consumption (VO2), dyspnoea and leg effort were measured in eight healthy humans (five men and three women, average age of 31 ± 4 years and body weight of 68 ± 10 kg), performing an incremental exercise testing on a cycle ergometer under control condition and expiratory flow limitation (FL) achieved by putting a Starling resistor. Blood samples were obtained at baseline, at peak of exercise and at iso-workload corresponding to that reached at the peak of FL exercise during control exercise. Diaphragmatic fatigue was evaluated by measuring the tension time index of the diaphragm. Respiratory muscle overloading caused an earlier interruption of exercise. Diaphragmatic fatigue did not occur in the two conditions. At peak of flow-limited exercise compared to iso-workload, HR, peak inspiratory and expiratory Pes, NK cells and norepinephrine were significantly higher. The number of NK cells was significantly related to ΔPes (i.e. difference between the most and the less negative Pes) and plasmatic catecholamines. Loading of respiratory muscles is able to cause an increase of NK cells provided that activation of respiratory muscles is intense enough to induce a significant metabolic stress.

Progressive hypoventilation due to mixed CD8+ and CD4+ lymphocytic polymyositis following tremelimumab - durvalumab treatment.

BACKGROUND: The combination of CTLA-4 and PD-L1 inhibitors has a manageable adverse effect profile, although rare immune-related adverse events (irAE) can occur. CASE PRESENTATION: We describe an autoimmune polymyositis following a partial response to combination tremelimumab and durvalumab for the treatment of recurrent lung adenocarcinoma. Radiography revealed significant reduction in all metastases; however, the patient developed progressive neuromuscular hypoventilation due to lymphocytic destruction of the diaphragmatic musculature. Serologic testing revealed a low level of de novo circulating antibodies against striated muscle fiber. Immunohistochemistry revealed type II muscle fiber atrophy with a mixed CD8+ and CD4+ lymphocyte infiltrate, indicative of inflammatory myopathy. CONCLUSIONS: This case supports the hypothesis that muscle tissue is a target for lymphocytic infiltration in immune checkpoint inhibitor-associated polymyositis. Further insights into the autoimmune mechanism of PM will hopefully contribute to the prevention and treatment of this phenomenon.

Non-invasive ventilation abolishes the IL-6 response to exercise in muscle-wasted COPD patients: a pilot study.

Systemic inflammation in patients with chronic obstructive pulmonary disease (COPD) has been related to the development of comorbidities. The level of systemic inflammatory mediators is aggravated as a response to exercise in these patients. The aim of this study was to investigate whether unloading of the respiratory muscles attenuates the inflammatory response to exercise in COPD patients. In a cross-over design, eight muscle-wasted stable COPD patients performed 40 W constant work-rate cycle exercise with and without non-invasive ventilation support (NIV vs control). Patients exercised until symptom limitation for maximally 20 min. Blood samples were taken at rest and at isotime or immediately after exercise. Duration of control and NIV-supported exercise was similar, both 12.9 ± 2.8 min. Interleukin- 6 (IL-6) plasma levels increased significantly by 25 ± 9% in response to control exercise, but not in response to NIV-supported exercise. Leukocyte concentrations increased similarly after control and NIV-supported exercise by ∼15%. Plasma concentrations of C-reactive protein, carbonylated proteins, and production of reactive oxygen species by blood cells were not affected by both exercise modes. This study demonstrates that NIV abolishes the IL-6 response to exercise in muscle-wasted patients with COPD. These data suggest that the respiratory muscles contribute to exercise-induced IL-6 release in these patients.

Elevated concentration of oxidized LDL together with poor cardiorespiratory and abdominal muscle fitness predicts metabolic syndrome in young men.

BACKGROUND: Metabolic syndrome (MetS) is associated with increased oxidized LDL (ox-LDL), systemic inflammation, and poor cardiorespiratory fitness. We examined affiliations of these factors and the effect of muscular fitness on MetS in young healthy men. METHODS: Physical fitness, ox-LDL, tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and serum lipids were measured in a nationally representative sample of Finnish young men with and without MetS. Participants (mean age 25.1years) performed tests of maximal oxygen uptake (VO2max) and muscle fitness, and were divided into MetS (n=54, IDF 2007 criteria) and non-MetS (n=790). Age, smoking and leisure-time physical activity were used as covariates (ANCOVA). RESULTS: The MetS group had lower results in VO2max and all of the muscular fitness tests (excluding grip strength) (P<0.0001, in all). Ox-LDL, ox-LDL/HDL-cholesterol, ox-LDL/LDL-cholesterol, TNFα and IL-6 were all higher in the MetS group than in the non-MetS group (P<0.01, in all). In stepwise multivariate logistic regression analysis (adjusted to MetS criteria), higher ox-LDL (OR 1.118, 95% CI 1.078-1.160), lower VO2max (OR 0.938, 95% CI 0.901-0.977) and lower sit-ups (OR 0.898, 95% CI 0.844-0.956) predicted MetS (p<0.05, in all). CONCLUSIONS: Young men with MetS possess significantly poorer cardiorespiratory and muscle fitness, together with elevated systemic levels of ox-LDL, TNFα and IL-6 compared to non-MetS young men. Of these variables, ox-LDL, VO2max and sit-ups predicted MetS. Based on these findings, poor physical fitness and elevated concentration of ox-LDL are significant predisposing factors in the development of MetS.

[Cytokines and resistive breathing].

This review summarizes and analyzes the results of the present experimental studies indicating immune system involvement in control of breathing. The hypothesis about the role of cytokines in the mechanisms of respiratory muscle fatigue and reduced ventilatory sensitivity to hypercapnia during respiration with the added resistive loading is justified. The possible ways of implementing of respiratory cytokine effects are discussed.

Vitamin D modulates airway smooth muscle function in COPD.

COPD is a disease manifested as persistent airflow obstruction with an enhanced inflammatory response in the airways and lungs to noxious particles and gases which evokes symptoms of dyspnea on exertion, cough and mucus production. Airway smooth muscle plays a central role in the COPD diathesis and is implicated in many aspects of COPD pathogenesis. Vitamin D deficiency has been associated with COPD severity and studies suggest a role for Vitamin D as a treatment for COPD. In this review, we describe the effects of 1,25-dihydroxyvitamin D on airway smooth muscle function, including agonist-induced shortening, secretion of inflammatory mediators, and myocyte hypertrophy and hyperplasia.

Inflammatory cells and apoptosis in respiratory and limb muscles of patients with COPD.

Discrepancies exist regarding the involvement of cellular inflammation and apoptosis in the muscle dysfunction of chronic obstructive pulmonary disease (COPD) patients with preserved body composition. We explored whether levels of inflammatory cells and apoptosis were increased in both respiratory and limb muscles of COPD patients without nutritional abnormalities. In the vastus lateralis, external intercostals, and diaphragms of severe and moderate COPD patients with normal body composition, and in healthy subjects, intramuscular leukocytes and macrophage levels were determined (immunohistochemistry). Muscle structure was also evaluated. In the diaphragm and vastus lateralis of severe and moderate COPD patients and controls, apoptotic nuclei were explored using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electron microscopy, and caspase-3 expression. In COPD patients compared with controls, diaphragm and intercostal levels of inflammatory cells were extremely low and not significantly different. However, in the vastus lateralis of the severe patients, inflammatory cell counts, although also very low, were significantly greater. In those patients, TUNEL-positive nuclei levels were also significantly greater in diaphragms and vastus lateralis. A significant inverse relationship was found between quadriceps TUNEL-positive nuclei levels and muscle force. Ultrastructural apoptotic nuclei revealed no differences in respiratory or limb muscles between COPD patients and controls. Muscle caspase-3 expression did not differ between patients and controls. In severe COPD patients with preserved body composition, while increased apoptotic nuclei seems to be a contributor to their muscle dysfunction, cellular inflammation does not. The increased numbers of TUNEL-positive nuclei in their muscles suggest that they may also be exposed to a continuous repair/remodeling process.

Agonists of toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes.

RATIONALE: Toll-like receptors 2 and 4 (TLR2, TLR4) enable cellular responses to bacterial lipoproteins, LPS, and endogenous mediators of cell damage. They have an established role in the activation of leukocytes, endothelial cells, and some smooth muscle cell types, but their roles in airway smooth muscle are uncertain. OBJECTIVES: To determine the roles of TLRs in activation of airway smooth muscle. METHODS: Airway smooth muscle cells were cultured with TLR agonists, in the presence or absence of mononuclear leukocytes. MEASUREMENTS AND MAIN RESULTS: We observed expression of TLR2 and TLR4 mRNAs, which could be upregulated by treatment with proinflammatory cytokines in primary human airway smooth muscle, but no important functional responses to agonists of these TLRs were seen. Coincubation of airway smooth muscle with peripheral blood mononuclear cells, at concentrations as low as 250 mononuclear cells/ml, resulted in a marked cooperative response to TLR stimuli, and synergistic production of cytokines, including chemokines (interleukin [IL-]-8) and IL-6. This cooperative response was greater when monocytes were enriched and was transferable using supernatants from LPS-stimulated peripheral blood mononuclear cells. Activation of cocultures required IL-1 generation from mononuclear cells, and was blocked by IL-1 receptor antagonist, though IL-1 generation alone was not sufficient to account for the magnitude of mononuclear cell-dependent coculture activation. CONCLUSIONS: These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.

Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen.

BACKGROUND: Airway smooth muscle hypertrophy is closely associated with the pathophysiology of hyper-reactive airways in allergic asthma. OBJECTIVE: To determine whether repeated exposure to allergens during postnatal lung development promotes remodelling of airway smooth muscle. METHODS: Infant, male rhesus monkeys (30-day-old) were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol periodically over 5 months. Smooth muscle mass and bundle size and abundance in conducting airways were measured and compared with age-matched control (filtered air-exposed) monkeys. RESULTS: Total smooth muscle mass and average bundle size were significantly greater in the conducting airways of monkeys exposed to HDMA. Smooth muscle bundle abundance was not affected by exposure to HDMA. CONCLUSION: Repeated cycles of allergen exposure alter postnatal morphogenesis of smooth muscle, affecting both total mass and bundle size, in conducting airways of infant monkeys.

Differential cytokine gene expression in the diaphragm in response to strenuous resistive breathing.

Strenuous resistive breathing induces plasma cytokines that do not originate from circulating monocytes. We hypothesized that cytokine production is induced inside the diaphragm in response to resistive loading. Anesthetized, tracheostomized, spontaneously breathing Sprague-Dawley rats were subjected to 1, 3, or 6 hours of inspiratory resistive loading, corresponding to 45-50% of the maximum inspiratory pressure. Unloaded sham-operated rats breathing spontaneously served as control animals. The diaphragm and the gastrocnemius muscles were excised at the end of the loading period, and messenger ribonucleic acid expression of tumor necrosis factor-alpha, tumor necrosis factor-beta, interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, and two housekeeping genes was analyzed using multiprobe RNase protection assay. IL-6, IL-1beta, and, to lesser extents, tumor necrosis factor-alpha, IL-10, IFN-gamma, and IL-4 were significantly increased in a time-dependent fashion in the diaphragms but not the gastrocnemius of loaded animals or in the diaphragm of control animals. Elevation of protein levels of IL-6 and IL-1beta in the diaphragm of loaded animals was confirmed with immunoblotting. Immunostaining revealed IL-6 protein localization inside diaphragmatic muscle fibers. We conclude that increased ventilatory muscle activity during resistive loading induces differential elevation of proinflammatory and antiinflammatory cytokine gene expression in the ventilatory muscles.

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation.

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.

Interferon-gamma modulates cysteinyl leukotriene receptor-1 expression and function in human airway myocytes.

Leukotrienes play a critical role in promoting bronchoconstriction in asthma. The purpose of this study was to examine whether interferon (IFN)-gamma, a cytokine upregulated in asthmatic airways, modulates leukotriene (LT)D4 receptor expression and contractile responses in cultured human airway smooth muscle (HASM) cells. Treatment of HASM cells with IFN-gamma (10 to 1,000 U/ml) stimulated a dose-dependent increase in cell-surface expression of cysteinyl leukotriene receptor 1 (CysLT1) as determined by flow cytometry. CysLT1 messenger RNA (mRNA) levels were also significantly enhanced by IFN-gamma, as demonstrated by reverse transcription-polymerase chain reaction. To determine the functional relevance of increased CysLT1 expression in HASM, cell stiffness responses to LTD4 were measured with magnetic twisting cytometry. IFN-gamma (1,000 U/ml for 24 h) markedly increased LTD4-induced changes in cell stiffness, from 4.6 +/- 1 [mean +/- SEM]% to 24.4 +/- 3.7% (n = 8, p < 0.05). Montelukast, a CysLT1 antagonist, completely inhibited LTD4-induced increases in cell stiffness. IFN-gamma had no effect on the cell stiffness responses to bradykinin, another contractile agonist. Collectively, these data suggest that IFN-gamma increases LTD4 responses in HASM cells by increasing cell-surface expression of CysLT1. Our data suggest that increased levels of IFN-gamma in asthmatic individuals may promote airway hyperresponsiveness and asthma exacerbations by directly modulating contractile responses of HASM.

Effect of endotoxin on ventilation and breath variability: role of cyclooxygenase pathway.

To evaluate the effects of endotoxemia on respiratory controller function, 12 subjects were randomized to receive endotoxin or saline; six also received ibuprofen, a cyclooxygenase inhibitor, and six received placebo. Administration of endotoxin produced fever, increased respiratory frequency, decreased inspiratory time, and widened alveolar-arterial oxygen tension gradient (all p < or = 0.001); these responses were blocked by ibuprofen. Independent of ibuprofen, endotoxin produced dyspnea, and it increased fractional inspiratory time, minute ventilation, and mean inspiratory flow (all p < or = 0.025). Endotoxin altered the autocorrelative behavior of respiratory frequency by increasing its autocorrelation coefficient at a lag of one breath, the number of breath lags with significant serial correlations, and its correlated fraction (all p < 0.05); these responses were blocked by ibuprofen. Changes in correlated behavior of respiratory frequency were related to changes in arterial carbon dioxide tension (r = 0.86; p < 0.03). Endotoxin decreased the oscillatory fraction of inspiratory time in both the placebo (p < 0.05) and ibuprofen groups (p = 0.06). In conclusion, endotoxin produced increases in respiratory motor output and dyspnea independent of fever and symptoms, and it curtailed the freedom to vary respiratory timing-a response that appears to be mediated by the cyclooxygenase pathway.

A comparative study on cockroach and ovalbumin sensitizations and challenge responses in Hartley guinea-pigs.

The role of allergens in asthmatic inflammation is not clearly understood. To elucidate the mechanism of cockroach allergen (CRa)-induced airway disease, we studied three groups of Hartley guinea-pigs sensitized to control, ovalbumin (OA) or CRa. Parameters measured were anaphylactic antibodies by allergy skin test (AST), PCA assay and Western blot, changes in specific airway resistance (SRaw), analysis of bronchoalveolar lavage (BAL) and contracture responses of tracheal muscle (TSM) to non-specific and specific stimuli, in vitro. Both OA and CRa animals showed a similar allergic sensitization (AST and PCA), while Western blot identified several reaginic bands in CRa group compared to a single band in OA group. SRaw illustrated that CRa induce dual-asthmatic responses (4/6) in the CRa group, whereas OA induce only an early asthmatic response (3/6) in the OA group (P<0.01). The average total leukocytes in BALF of the CRa were 27.0x10(6), mostly neutrophils and eosinophils, while those of the OA showed 3.5x10(6), mostly eosinophils, respectively (P<0.0001). TSM responses to non-specific stimuli were similar in both groups (P>0.1), while the antigen-specific TSM contractions were more brisk in the OA group than those of CRa group (P<0.001). Thus, the study indicates that both CRa and OA sensitize guinea-pigs, yet CRa induces more severe and persistent late-phase inflammation than OA. This appears to be related to an influx of neutrophils rather than anaphylactic bronchospasm.

[The effect of sensitization to the electrical activity of the muscles of the lower respiratory tract in the rat]. / Vliianie sensibilizatsii na élektricheskuiu aktivnost' myshts nizhnikh dykhatel'nykh putei krysy.

The electrical activity of the smooth muscle of the trachea was studied on sensibilized ovalbumine rats. The role of intramural ganglions of the airway was shown.