Paradoxical effects of cigarette smoke and COPD on SARS-CoV-2 infection and disease.

BACKGROUND: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro. METHODS: We quantified: (1) pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and/or TMPRSS2 mRNA levels by RT-qPCR in two independent human cohorts; and (2) pulmonary ACE2 protein levels by immunostaining and ELISA in C57BL/6 WT mice exposed to air or CS for up to 6 months. The effects of CS exposure on SARS-CoV-2 infection were evaluated after in vitro infection of Calu-3 cells and differentiated human bronchial epithelial cells (HBECs), respectively. RESULTS: ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus controls but similar in central airways. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice. CS treatment decreased viral replication in Calu-3 cells, as determined by immunofluorescence staining for replicative double-stranded RNA (dsRNA) and western blot for viral N protein. Acute CS exposure decreased in vitro SARS-CoV-2 replication in HBECs, as determined by plaque assay and RT-qPCR. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-exposure potently inhibited SARS-CoV-2 replication in vitro. These findings urge to investigate further the controversial effects of CS and COPD on SARS-CoV-2 infection.

Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease.

INTRODUCTION: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. METHODS: In Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. RESULTS: In both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.

Muscarinic inhibition of two potassium currents in guinea-pig prevertebral neurons: differentiation by extracellular cesium.

Muscarinic responses were studied in dissociated guinea-pig celiac ganglion neurons using the whole-cell voltage-clamp technique. Muscarine (0.025-1 mM; EC50 = 95 microM) administered to cells for 1.5 s evoked inward shifts in holding current in 53 of 74 cells. The amplitude of the inward current transients decreased with hyperpolarization and the null potential averaged -71 +/- 3.4 mV (n = 11). The currents that underlie the responses to muscarine were examined with hyperpolarizing voltage stepping protocols to -100 mV from a holding potential of -30 mV. Eighty-one per cent of cells displayed voltage-dependent current relaxations characteristic of the M-potassium current. Twenty per cent of responding cells displayed no M-current but only a voltage-independent current consistent with a leak current. In the latter type of cells, the muscarine-evoked inward currents reversed near EK and became outward at more hyperpolarized potentials. Analysis of steady state I-V relationships before and after bath application of muscarine showed that the two muscarine-sensitive potassium currents were distributed differently among three types of cells: (i) with M-current (18%); (ii) with leak current (18%); and (iii) with M-current and with leak current (64%). Cesium and barium were used to differentiate the M-current and the muscarine-sensitive leak current. Barium (2 mM) reduced the M-current and the leak potassium current, whereas cesium (2 mM) reduced the M-current but did not affect leak current. Thus, barium reduced the amplitude of muscarinic responses by 79% but cesium reduced them by only 14%. We conclude that muscarinic responses in guinea-pig celiac neurons are produced by suppression of two K+ currents: the M-current and a muscarine-sensitive leak current. These two currents are differentially susceptible to the potassium channel blockers barium and cesium.

Systemic fungal infections: diagnosis and treatment. I. Coccidioidomycosis.

Coccidioidomycosis is a highly variable disease. Initial respiratory tract infection can lead to self-limited pneumonia, pulmonary complications, and extrapulmonary disease. The early infection requires no therapy, except in immunosuppressed patients and other selected patients. Treatment for pulmonary complications may include surgery for cavities or pyopneumothorax (resulting from rupture of a cavity) and antifungal therapy for chronic pneumonia. The majority of extrapulmonary disease occurs in the skin, bones and joints, or meninges and is an indication for treatment with antifungal agents and sometimes adjunctive surgery. Meningitis is a particularly serious consequence of dissemination and currently is best treated with intrathecal instillation of antifungal agents. Antifungal agents useful in the treatment of coccidioidomycosis are amphotericin B, which is administered intravenously and is relatively toxic, and ketoconazole, which is administered orally and whose toxicities are less serious and reversible. Because studies to compare the efficacy of these two drugs have not been performed, selecting between them for use in individual patients is most rationally based on the pharmacologic differences, which lend themselves to different clinical settings. In future years, new antifungal agents will likely be available, some of which will offer significant advantages over present therapies. Itraconazole is an imidazole related to ketoconazole, which appears to be effective and possibly less toxic than ketoconazole. Fluconazole, another imidazole, has broad antifungal activity, a long serum half-life, and excellent penetration into the cerebrospinal fluid. Thus, the pharmacology of this agent would appear ideal for use in treating coccidioidal meningitis. In addition, other compounds with different modes of action are now under investigation in preclinical studies. It is therefore likely that continued improvements will occur in the coming years in the treatment of this disease.

The muscarinic receptor agonist oxotremorine methiodide evokes a nicotinic response in mammalian sympathetic neurons.

Oxotremorine methiodide, a congener of oxotremorine, is used as a muscarinic receptor agonist. Responses to oxotremorine methiodide and nicotinic receptor agonists were examined in cultured guinea-pig celiac ganglion neurons using whole-cell voltage clamp techniques. At holding potentials between -30 and -60 mV, a brief application of oxotremorine methiodide produced fast and slow inward current transients, depending upon the concentration applied. Slowly developing inward current transients, characteristic of muscarinic responses, were produced by lower concentrations (EC50: 0.3 microM) and were blocked by atropine. Rapid inward current transients, characteristic of nicotinic responses, were produced by higher concentrations of oxotremorine methiodide (EC50: 168 microM) and were blocked by d-tubocurarine. Thus oxotremorine methiodide, at concentrations of 10 microM and greater, produced an initial nicotinic fast inward current transient followed by a slow muscarinic inward transient. The fast inward transients were similar to responses evoked by the nicotinic receptor agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide and were not antagonized by atropine. We conclude that oxotremorine methiodide acts as a nicotinic and muscarinic receptor agonist in celiac sympathetic ganglion neurons.

CCKA receptors mediate slow depolarizations in cultured mammalian sympathetic neurons.

The effect of cholecystokinin octapeptide (CCK-8) was examined in guinea-pig celiac ganglion (CG) neurons in primary culture using standard intracellular recording techniques. Sulfated CCK-8 (CCK-8S; 1 microM) evoked slow depolarizing responses in 94% of CG neurons tested. In contrast, membrane potential was not affected by nonsulfated CCK-8 (CCK-8NS; 1 microM), CCK tetrapeptide (CCK-4; 1 microM), or gastrin (1 microM). The selective CCKA receptor antagonist L 364,718 potently inhibited CCK-8S-induced slow depolarizations (IC50 2.9 pM). In contrast, the selective CCKB receptor antagonist L 365,260 was a weak inhibitor of CCK-8S-induced slow depolarizations (IC50 1.3 microM). The depolarizing responses to CCK-8S were associated with an average increase in cell input resistance of 61%. Single electrode voltage clamp experiments indicated that CCK-8S-induced depolarizations were associated with a slow inward shift in holding current. Thus, the present findings indicate that guinea-pig cultured CG neurons are endowed with excitatory CCKA receptors the activation of which elicits a decrease in membrane conductance, thereby resulting in slow depolarizations.

Response to 5-hydroxytryptamine on neurons of guinea pig celiac and inferior mesenteric ganglia in primary culture.

The electrophysiological effects of serotonin, a putative neurotransmitter in prevertebral sympathetic ganglia, were evaluated in cultured celiac and inferior mesenteric ganglia (IMG) neurons. Intracellular microelectrode recordings were performed in neurons that were maintained in culture an average of 26 days. Seventy-eight of 85 neurons responded when serotonin (10 microM) was applied by pressure ejection from a micropipette to the surface of the isolated cells. The majority of the neurons (n = 48) generated fast depolarizations, although slow depolarizations (n = 17), bipolar responses (n = 5), hyperpolarizations (n = 7), and a biphasic response (n = 1), were also seen. Hyperpolarizing responses were evoked in celiac neurons only. All responses were inhibited by the 5-HT3 antagonist MDL 72,222 (5 microM). Fast responses were not inhibited by tetrodotoxin (n = 3). These results demonstrate that serotonin evokes a variety of membrane potential changes in cultured prevertebral sympathetic neurons by activating 5-HT3 receptors.

Segmental wall motion abnormalities in an individual with idiopathic pulmonary hemosiderosis.

Idiopathic pulmonary hemosiderosis (IPH) is a rare condition characterized by diffuse pulmonary hemorrhage of unknown etiology. Cardiac involvement in the form of myocarditis and right ventricular hypertrophy have been reported to occur in association with IPH, although findings on echocardiography have not been described. Herein is presented a case of an adult with IPH and echocardiographic abnormalities.