Co-infections and superinfections complicating COVID-19 in cancer patients: A multicentre, international study.

BACKGROUND: We aimed to describe the epidemiology, risk factors, and clinical outcomes of co-infections and superinfections in onco-hematological patients with COVID-19. METHODS: International, multicentre cohort study of cancer patients with COVID-19. All patients were included in the analysis of co-infections at diagnosis, while only patients admitted at least 48 h were included in the analysis of superinfections. RESULTS: 684 patients were included (384 with solid tumors and 300 with hematological malignancies). Co-infections and superinfections were documented in 7.8% (54/684) and 19.1% (113/590) of patients, respectively. Lower respiratory tract infections were the most frequent infectious complications, most often caused by Streptococcus pneumoniae and Pseudomonas aeruginosa. Only seven patients developed opportunistic infections. Compared to patients without infectious complications, those with infections had worse outcomes, with high rates of acute respiratory distress syndrome, intensive care unit (ICU) admission, and case-fatality rates. Neutropenia, ICU admission and high levels of C-reactive protein (CRP) were independent risk factors for infections. CONCLUSIONS: Infectious complications in cancer patients with COVID-19 were lower than expected, affecting mainly neutropenic patients with high levels of CRP and/or ICU admission. The rate of opportunistic infections was unexpectedly low. The use of empiric antimicrobials in cancer patients with COVID-19 needs to be optimized.

Hydrogen sulphide as a signalling molecule regulating physiopathological processes in gastrointestinal motility.

The biology of H2 S is a still developing area of research and several biological functions have been recently attributed to this gaseous molecule in many physiological systems, including the cardiovascular, urogenital, respiratory, digestive and central nervous system (CNS). H2 S exerts anti-inflammatory effects and can be considered an endogenous mediator with potential effects on gastrointestinal motility. During the last few years, we have investigated the role of H2 S as a regulator of gastrointestinal motility using both animal and human tissues. The aim of the present work is to review published data regarding the potential role of H2 S as a signalling molecule regulating physiopathological processes in gastrointestinal motor function. H2 S is endogenously produced by defined enzymic pathways in different cell types of the intestinal wall including neurons and smooth muscle. Inhibition of H2 S biosynthesis increases motility and H2 S donors cause smooth muscle relaxation and inhibition of propulsive motor patterns. Impaired H2 S production has been described in animal models with gastrointestinal motor dysfunction. The mechanism(s) of action underlying these effects may include several ion channels, although no specific receptor has been identified. At this time, even though there is much experimental evidence for H2 S as a modulator of gastrointestinal motility, we still do not have conclusive experimental evidence to definitively propose H2 S as an inhibitory neurotransmitter in the gastrointestinal tract, causing nerve-mediated relaxation.

Inverse gradient of nitrergic and purinergic inhibitory cotransmission in the mouse colon.

AIM: Gastrointestinal smooth muscle relaxation is accomplished by the neural corelease of ATP or a related purine and nitric oxide. Contractions are triggered by acetylcholine and tachykinins. The aim of this work was to study whether regional differences in neurotransmission could partially explain the varied physiological roles of each colonic area. METHODS: We used electrophysiological and myography techniques to evaluate purinergic (L-NNA 1 mm incubated tissue), nitrergic (MRS2500 0.3 µm incubated tissue) and cholinergic neurotransmission (L-NNA 1 mm and MRS2500 0.3 µm incubated tissue) in the proximal, mid and distal colon of CD1 mice (n = 42). RESULTS: Purinergic electrophysiological responses elicited by single pulses (28 V) were greater in the distal (IJPfMAX = -35.3 ± 2.2 mV), followed by the mid (IJPfMAX = -30.6 ± 1.0 mV) and proximal (IJPfMAX = -11.7 ± 1.1 mV) colon. In contrast, nitrergic responses decreased from the proximal colon (IJPsMAX = -11.4 ± 1.1 mV) to the mid (IJPsMAX = -9.1 ± 0.4 mV), followed by the distal colon (IJPsMAX = -1.8 ± 0.3 mV). A similar rank of order was observed in neural mediated inhibitory mechanical responses including electrical field stimulation-mediated responses and neural tone. ADPßs concentration-response curve was shifted to the left in the distal colon. In contrast, NaNP responses did not differ between regions. Cholinergic neurotransmission elicited contractions of a similar amplitude throughout the colon. CONCLUSION: An inverse gradient of purinergic and nitrergic neurotransmission exists through the mouse colon. The proximal and mid colon have a predominant nitrergic neurotransmission probably due to the fact that their storage function requires sustained relaxations. The distal colon, in contrast, has mainly purinergic neurotransmission responsible for the phasic relaxations needed to propel dehydrated faeces.

Potential role of the gaseous mediator hydrogen sulphide (H2S) in inhibition of human colonic contractility.

BACKGROUND: Hydrogen sulphide (H2S) is an endogenous signalling molecule that might play a physiologically relevant role in gastrointestinal motility. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are two enzymes responsible for H2S production. d,l-Propargylglycine (PAG) is a CSE inhibitor whereas both aminooxyacetic acid (AOAA) and hydroxylamine (HA) are CBS inhibitors. The characterization of H2S responses and its mechanism of action are crucial to define H2S function. METHODS: Human colonic strips were used to investigate the role of H2S on contractility (muscle bath) and smooth muscle electrophysiology (microelectrodes). NaHS was used as a H2S donor. RESULTS: Combination of PAG and AOAA depolarized the smooth muscle (5-6mV, n=4) and elicited a transient increase in tone (260.5±92.8mg, n=12). No effect was observed on neural mediated inhibitory junction potential or relaxation. In the presence of tetrodotoxin 1µM, NaHS concentration-dependently inhibited spontaneous contractions (EC50=329.2µM, n=18). This effect was partially reduced by the guanylyl cyclase inhibitor ODQ 10µM (EC50=2.6µM, n=12) and by l-NNA 1mM (EC50=1.4mM, n=8). NaHS reversibly blocked neural mediated cholinergic (EC50=2mM) and tachykinergic (EC50=5.7mM) contractions. NaHS concentration-dependently reduced the increase in spontaneous mechanical activity (AUC) induced by carbachol (EC50=1.9mM) and NKA (EC50=1.7mM AUC). CONCLUSIONS: H2S might be an endogenous gasomediator regulating human colonic contractility. Its inhibitory effect is observed at high concentrations and could be mediated by a direct effect on smooth muscle with a possible synergistic effect with NO, as well as by an interaction with the cholinergic and tachykinergic neural mediated pathways.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

BACKGROUND: Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. METHODS: Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. RESULTS: In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1µM) and EP4 receptor antagonists (L-161,982 10µM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1µM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10µM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. CONCLUSIONS: PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation.

AIM: ATP and nitric oxide (NO) are released from enteric inhibitory motor neurones and are responsible for colonic smooth muscle relaxation. However, how frequency of neural stimulation affects this cotransmission process and the post-junctional responses has not been systematically characterized in the human colon. METHODS: The dynamics of inhibitory cotransmission were studied using different protocols of electrical field stimulation (EFS) to characterize the inhibitory junction potentials (IJP) and the corresponding relaxation in colonic strips obtained from 36 patients. RESULTS: Single pulses elicited a fast IJP (IJPf(MAX) = -27.6 ± 1.6 mV), sensitive to the P2Y1 antagonist MRS2500 1 µm, that ran down with frequency increase leaving a residual hyperpolarization at high frequencies (IJPf∞ = -3.7 ± 0.6 mV). Accordingly, low frequencies of EFS caused purinergic transient relaxations that cannot be maintained at high frequencies. Addition of the P2Y1 agonist MRS2365 10 µm during the purinergic rundown did not cause any hyperpolarization. Protein kinase C (PKC), a putative P2Y1 desensitizator, was able to reduce the amplitude of the IJPf when activated, but the rundown was not modified by PKC inhibitors. Frequencies higher than 0.60 ± 0.15 Hz were needed to evoke a sustained nitrergic hyperpolarization that progressively increased reaching IJPs∞ = -13 ± 0.4 mV at high frequencies and leading to a sustained inhibition of spontaneous motility. CONCLUSION: Changes in frequency of stimulation possibly mimicking neuronal firing will post-junctionally determine purinergic vs. nitrergic responses underlying different functional roles. NO will be responsible for sustained relaxations needed in physiological processes such as storage, while purinergic neurotransmission evoking sharp transient relaxations will be dominant in processes such as propulsion.

Mechanisms of action of otilonium bromide (OB) in human cultured smooth muscle cells and rat colonic strips.

BACKGROUND: The pharmacological properties of otilonium bromide (OB) have been investigated using different experimental models, techniques, and conditions, and consequently, the results are not always easy to compare. The aim of the present work was to investigate the pharmacological properties of OB in human cultured colonic smooth muscle cells (HCSMCs), which is the main target of the drug 'in vivo'. Rat colonic strips were used to confirm the pharmacological properties. METHODS: Human cultured colonic smooth muscle cells were studied using the calcium imaging technique. Microelectrodes and muscle bath experiments were performed in rat colonic strips. KEY RESULTS: Otilonium bromide (OB) concentration dependently inhibited nifedipine-sensitive calcium transients induced by KCl (EC50  = 3.6 µM) and BayK8644 (EC50  = 4.0 µM). All the following experiments were performed in the presence of nifedipine. In HCSMC, carbachol-induced calcium transients were inhibited by OB (EC50  = 8.4 µM). Carbachol evoked 1-a smooth muscle depolarization (10 mV) that was antagonized by 100 µM OB; and 2-a contraction that was inhibited by OB (EC50  = 13.0 µM). 'Non-nitrergic (L-NNA 1 mM) non-purinergic (MRS2500 1 µM)' conditions were used to elicit endogenous excitatory responses. Electrical field stimulation caused 1-an atropine-sensitive excitatory junction potential that was inhibited by OB (EC50  = 8.9 µM) and 2-an atropine-sensitive contraction that was inhibited by OB (EC50  = 7.3 µM). In HCSMC, neurokinin A (NKA) and CaCl2 induced calcium transients that were inhibited by OB (NKA: EC50  = 11.7 µM; CaCl2 : EC50  = 17.5 µM). CONCLUSIONS & INFERENCES: Otilonium bromide causes inhibition of L-/T-type calcium channels, muscarinic, and tachykininergic responses that acting together explain the pharmacological properties of the compound.

P2Y(1) knockout mice lack purinergic neuromuscular transmission in the antrum and cecum.

BACKGROUND: Pharmacological studies using selective P2Y(1) antagonists, such as MRS2500, and studies with P2Y(1)(-/-) knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y(1) receptors in the colon. The aim of the present study was to test whether P2Y(1) receptors are involved in purinergic neurotransmission in the antrum and cecum. METHODS: Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y(1)(-/-) mice. KEY RESULTS: In the antrum, no differences in resting membrane potential and slow wave activity were observed between groups. In WT animals, electrical field stimulation elicited a MRS2500-sensitive inhibitory junction potential (IJP). In P2Y(1)(-/-) mice, a nitrergic IJP (N(ω) -nitro-l-arginine-sensitive), but not a purinergic IJP was recorded. This IJP was equivalent to the response obtained in strips from WT animals previously incubated with MRS2500. Similar results were obtained in the cecum: 1- the purinergic IJP (MRS2500-sensitive) recorded in WT animals was absent in P2Y(1)(-/-) mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y(1)(-/-) mice 2- MRS2365 a P2Y(1) agonist caused smooth muscle hyperpolarization in WT, but not in P2Y(1) (-/-) animals, and 3- ß-NAD caused smooth muscle hyperpolarization both in WT and P2Y(1)(-/-) animals. CONCLUSIONS & INFERENCES: 1- P2Y(1) receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y(1)(-/-) mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y(1)(-/-) mouse might help in the identification of purinergic neurotransmitter(s).

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon.

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 µM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 µM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 µM), ODQ (10 µM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 µM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 µM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 µM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

Pharmacological characterization of purinergic inhibitory neuromuscular transmission in the human colon.

BACKGROUND: In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y(1) receptor. The P2Y(12) antagonist AR-C66096 was tested as well. Using this pharmacological approach, we tested whether ß-nicotinamide adenine dinucleotide (ß-NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon. METHODS: We carried out muscle bath and microelectrode experiments on circular strips from the human colon and calcium imaging recordings on HEK293 cells, which constitutively express the human P2Y(1) receptor. KEY RESULTS: Both the fast component of IJP and non-nitrergic relaxation was concentration-dependently inhibited by MRS2279 and MRS2500. This antagonism was confirmed in HEK293 cells. However, AR-C66096 did not modify either inhibitory response. Adenosine 5'-Ο-2-thiodiphosphate and MRS2365 caused a smooth muscle hyperpolarization and transient inhibition of spontaneous motility that was antagonized by MRS2279 and MRS2500. ß-Nicotinamide adenine dinucleotide inhibited the spontaneous motility (IC(50) = 3.3 mmol L(-1) ). Nevertheless, this effect was not antagonized by high concentrations of P2Y(1) antagonists. CONCLUSIONS & INFERENCES: Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y(1) receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y(1) antagonists is MRS2500 > MRS2279 > MRS2179. We found that ß-NAD partially fulfills the criteria to be considered an inhibitory neurotransmitter in the human colon, but the relative contribution of each purine (ATP/ADP vsß-NAD) requires further studies.