Immunomodulatory agents as potential therapeutic or preventive strategies for COVID-19.

Currently, the COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, represents the greatest global health threat. Most people infected by the virus present mild to moderate respiratory symptoms and recover with supportive treatments. However, certain susceptible hosts develop an acute respiratory distress syndrome (ARDS), associated with an inflammatory "cytokine storm", leading to lung damage. Despite the current availability of different COVID-19 vaccines, the new emerging SARS-CoV-2 genetic variants represent a major concern worldwide, due to their increased transmissibility and rapid spread. Indeed, it seems that some mutations or combinations of mutations might confer selective advantages to the virus, such as the ability to evade the host immune responses elicited by COVID-19 vaccines. Several therapeutic approaches have been investigated but, to date, a unique and fully effective therapeutic protocol has not yet been achieved. In addition, steroid-based therapies, aimed to reduce inflammation in patients with severe COVID-19 disease, may increase the risk of opportunistic infections, increasing the hospitalization time and mortality rate of these patients. Hence, there is an unmet need to develop more effective therapeutic options. Here, we discuss the potential use of natural immunomodulators such as Thymosin α1 (Tα1), all-trans retinoic acid (ATRA), and lactoferrin (LF), as adjunctive or preventive treatment of severe COVID-19 disease. These agents are considered to be multifunctional molecules because of their ability to enhance antiviral host immunity and restore the immune balance, depending on the host immune status. Furthermore, they are able to exert a broad-spectrum antimicrobial activity by means of direct interactions with cellular or molecular targets of pathogens or indirectly by increasing the host immune response. Thus, due to the aforementioned properties, these agents might have a great potential in a clinical setting, not only to counteract SARS-CoV-2 infection, but also to prevent opportunistic infections in critically ill COVID-19 patients.

Antimicrobial properties of the medicinal plant Cardiospermum halicacabum L: new evidence and future perspectives.

The emergence and rapid spread of multidrug-resistance in human pathogenic microorganisms urgently require the development of novel therapeutic strategies for the treatment of infectious diseases. From this perspective, the antimicrobial properties of the natural plant-derived products may represent an important alternative therapeutic option to synthetic drugs. Among medicinal plants, the Cardiospermum halicacabum L. (C. halicacabum), belonging to Sapindaceae family, could be a very promising candidate for its antimicrobial activity against a wide range of microorganisms, including both Gram-positive and Gram-negative bacteria, as well as fungal pathogens. Although the antimicrobial properties of C. halicacabum have been intensively studied, the mechanism/s by which it exerts the inhibitory activity towards the pathogenic microbes have not yet been completely understood. This review focuses on the main antimicrobial activities displayed in vitro by the plant extract, with particular attention on our recent advances. We demonstrated that C. halicacabum is able to exert in vitro a dose-dependent fungistatic effect against Trychophyton rubrum (T. rubrum) through molecular interaction with the fungal heat shock protein (Hsp)-90 chaperone. These findings are supported by a growing body of research indicating the crucial role played by the Hsp90 in the virulence of the pathogenic microorganisms, including fungal pathogens. The possible future use of C. halicacabum for treating a wide range of infectious diseases is also discussed.

Evaluation of the antifungal effect of EDTA, a metal chelator agent, on Candida albicans biofilm.

OBJECTIVE: Candida albicans biofilm is frequently found on artificial surfaces and the infections related to biofilm are difficult to eliminate, as they require the removal of artificial devices and treatment with antifungal drugs. Nowadays, fungal growth in biofilms is difficult to eradicate with conventional antifungal drugs such as fluconazole. Among chelating agents, disodium salt-Ethylene Diamine Tetraacetic Acid (EDTA) is known to have antifungal activity. In this study, we examined the in vitro activity of the EDTA and the antifungal drug fluconazole against C. albicans mature biofilm. MATERIALS AND METHODS: C. albicans ATCC 20191, fluconazole-susceptible strain, was grown at an inoculum starter of 1 x 106 cells/ml for 72 h in 24-well microtiter plates and was further treated for 24 h with EDTA and/or fluconazole. Antifungal activities in biofilms were expressed as reduction in optical density (OD) determined by a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay and compared to untreated biofilms. RESULTS: Colorimetric readings revealed that EDTA alone (at 25 and 2.5 mM) significantly reduced fungal metabolic activity in preformed biofilms. Also, EDTA combined with fluconazole significantly reduced the growth of biofilm when compared to biofilm treated with fluconazole alone (at 25 and 2.5 µg/ml). CONCLUSIONS: Our data suggest that the employment of EDTA or other chemicals destabilizers of the biofilm matrix, in combination with antifungal drugs, could lead to the development of new strategies for the management of infections associated to Candida biofilm. Another relevant result of our study suggests that the initial cell concentration, probably through mechanisms of quorum sensing, affects the cellular viability during the process of biofilm formation.

(1-3)-ß-D-Glucan vs Galactomannan Antigen in Diagnosing Invasive Fungal Infections (IFIs).

Invasive fungal infections (IFIs) are serious and often life-threatening complications in patients with haematological malignancies. Early diagnosis and the initiation of efficacious antifungal treatments could affect the prognosis of these patients. The detection of (1-3)-ß-D-Glucan (BDG) could be a promising non-culture-based, noninvasive tool for IFI analyses in haemato-oncological patients, allowing the diagnosis of the two major IFIs, invasive aspergillosis (IA) and invasive candidiasis (IC), with a single test. The aim of this work was to evaluate and compare the use of the BDG in combination with the galactomannan antigen (GAL) assay in order to exclude or confirm suspected IFIs. Sera from 46 haemato-oncological patients (24 with proven/probable IFI and 22 without IFI symptoms) were evaluated retrospectively for the detection of GAL and BDG. In 24 patients, the serum BDG levels facilitated IFI diagnosis: 18 probable IA, 3 proven IA and 3 IC. In the remaining 22 patients, the BDG level helped exclude IFIs. The BDG was positive earlier than GAL in 5/24 cases [three of probable invasive aspergillosis (IA), one of proven IA and one case of proven invasive candidiasis (IC)] and was positive at the same time as GAL in 19/24 cases; in no case was GAL positive before BDG was. The BDG detection is useful, however, the test has a great limitation because it is a completely manual procedure.

Effect of morphine on cell-mediated immune responses of human lymphocytes against allogeneic malignant cells.

Opioid drugs, including morphine, are largely used as pain control in cancer patients at different stages of neoplastic growth and progression. Therefore, the possible influence of these drugs on host immunity appears to be of considerable interest. We have examined in vitro the effect of morphine on the generation of human cytotoxic T lymphocytes (CTL) against HTLV-I induced T-cell leukemia cells (MT-2 line). The results show that the drug, at graded concentrations (from 3 pg/ml to 32 microg/ml), that include those detectable in treated patients, enhances CTL activity whereas natural killer cell activity was unaffected. The enhancing effect is particularly evident when morphine was present at the onset of lymphocyte/MT-2 co-culture. On the contrary, the drug was ineffective when added on the last day of co-culture, thus indicating that morphine operates during the generation phase of CTL, but not on mature CTL. Flow cytometric analysis of intracellular cytokine expression showed that morphine increases the percentage of interferon gamma-producing CD8+ T cells in co-culture assay. Collectively, these results suggest that in our experimental model morphine enhances CTL responses by directly affecting the induction phase of T-dependent cell-mediated immunity.

Fluconazole resistance in Candida albicans: a review of mechanisms.

Antifungal agents have greatly contributed to the improvement of public health. Nevertheless, antifungal resistant pathogens have increased during the past decade, becoming a serious concern. Candida albicans has been the most extensively studied pathogen in antifungal resistance because of their morbidity and mortality associated with infections in immunocompromised patients. This review describes the antifungal mechanims of the azole fluconazole widely used for the prophylaxis and treatment of candidal infections. The specific molecular pathways occurring in fluconazole-resistance of C. albicans and some issues about new antifungal agents are also discussed.

Cytokine pattern secretion by murine spleen cells after inactivated Candida albicans immunization. Effect of cocaine and morphine treatment.

In the present work we have analyzed: i) the effect of heat-inactivated Candida albicans immunization on the cytokine production by murine spleen cells; ii) the effect of a subchronic cocaine and morphine treatment on this production. The treatment with a single dose of inactivated Candida blastospores induced interleukin-2(IL-2), interferon-gamma (IFNgamma) and interleukin-4 (IL-4) production at 24 h after in vitro restimulation of splenocytes. In this model, the exposure to morphine (25 mg/kg, 5 days before, during and 5 days after inoculation with the yeast) significant decreased IL-2 and IL-4 levels, while secretion of IFN-gamma was unaltered. The same cocaine treatment (10 mg/kg) resulted in unchanged levels of the three cytokines tested. The results showed that non-viable Candida cells of this strain induce a predominant Th0 response. This immune effect is in part impaired only by a subchronic administration of morphine.

Maternal morphine alters parvalbumin immunoreactivity patterns in neonatal mouse brain.

The influence of chronic maternal morphine on the parvalbumin immunoreactive patterns in developing mouse brain was studied. Female Swiss mice were administered daily saline or morphine (30 or 60 mg/kg) for a period of 7 days before mating, gestation, and 21 days postpartum. Their pups were sacrificed on postnatal day 18 and the brains were examined histologically and immunohistochemically for parvalbumin-positive neurons. Histological observations revealed no significant changes in the cell number of the morphine-exposed neonatal forebrain, whereas the number of parvalbumin-positive neurons increased in layers II-IV of the parietal cortex I. Moreover, the number of parvalbumin-positive dendrites increased remarkably in the cingulate and parietal I cortices of the morphine-exposed neonates, indicating the region-specific increase in the PV immunoreactive profiles. These results are consistent with the key roles played by the above brain regions in the altered behavioral patterns of the maternally addicted neonates, such as impaired somatosensory and cognitive performances. The mechanism of morphine action on parvalbumin expression in neonatal mouse brain is not evident, but alterations in the expression patterns of parvalbumin in specific regions of the developing brain might be one of the cellular mechanisms by which addictive drugs modify the functional aspects of the developing CNS.

Effects of cocaine administration to influenza virus-immunized mice on cytokine profiles of individual splenic CD4+ and CD8+ T cells.

We have analysed the effects of cocaine, administered to mice during the in vivo differentiation of effector T cells stimulated by antigen (influenza virus) recognition, on the frequency of IL-2-, IL-4- and interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ T cells. Each animal was injected intraperitoneally with 10 mg/kg of cocaine 6, 24, 48 and 72 h after immunization with A/PR8 influenza virus (PR8). This enabled the determination of the pharmacological effects of cocaine on T cells during the initial step of the immune response, which is characterized by the production of large amounts of immunoregulatory cytokines. The distribution of IL-2-, IL-4- and IFN-gamma-producing CD4+ and CD8+ T cells was assayed on unseparated PR8-immune spleen cells, obtained from mice treated with cocaine or vehicle, and restimulated in vitro with UV-inactivated PR8 virus. The frequency of T cells singly or co-expressing the above three cytokines was determined at single-cell level by simultaneous flow cytometric analysis of intracellular cytokines and surface antigen expression. In parallel, the levels of IL-2, IL-4 and IFN-gamma in the culture supernatants were quantified by ELISA. The results showed that cocaine, administered during the in vivo virus-induced differentiation of T cells, caused an increase of both the frequencies of CD8+ T cells singly and co-expressing IL-2 and IFN-gamma and the levels of these cytokines in virus-restimulated spleen cell culture supernatants, compared with those of untreated controls. In contrast, no effect was found on IL-4-positive CD8+ T cells and on IL-2-, IFN-gamma- and IL-4-positive CD4+ T cells. Our findings suggest that the immunomodulatory effects of cocaine may be due to the up-regulation of the production of IL-2 and IFN-gamma by CD8+ T cells with a type 0 cytokine profile.

Cocaine potentiates the switch between latency and replication of Epstein-Barr virus in Raji cells.

This paper shows that cocaine amplifies Epstein-Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca(2+) mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein-Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.

Increased replication of Sendai virus in morphine-treated epithelial cells: evidence for the involvement of the intracellular levels of glutathione.

This paper shows that morphine increases Sendai virus replication in cultured epithelial cells. The effect was maximal when it was added before viral infection. Morphine also reduced the intracellular level of glutathione, namely, the oxidative and most abundant cell thiol. Altered intracellular redox status has recently been proposed as a factor influencing viral infection. Support for this view was provided by our data showing that inhibition of de novo glutathione synthesis, using L-buthionine sulfoximine, increased virus replication. These findings provide the first evidence that morphine increases the susceptibility to virus infection by altering the intracellular levels of glutathione.

Cocaine induced T cell proliferation in the rat: role of amygdala dopamine D1 receptors.

The immunomodulatory effects of local administration of cocaine into the amygdala were studied in the rat. Intra-amygdala infusion of cocaine significantly and dose-dependently increased the proliferative response of splenocytes to concanavalin A (Con A). A similar effect on the immune response was also observed in rats, microinfused into the central amygdala with the selective D1 receptor agonist SKF 38393. The increase of the proliferative response of splenocytes to Con A was inhibited by coinfusion within the central amygdala of the dopamine D1 receptor antagonist SCH 23390, together with cocaine, but not by coinfusion of the dopamine D2 receptor antagonist eticlopride. These results suggest that cocaine may produce at least some of its effects on the immune system through the activation of brain dopamine neurotransmission and that the central amygdala may represent a critical structure mediating cocaine-induced T cell proliferation.

Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains.

This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.

Chronic morphine alters calbindin D-28k immunostaining patterns in mouse forebrain.

The influence of chronic morphine treatment on the brain of adult mouse has been studied. Female Swiss mice were daily administered saline or morphine (30 or 60 mg/kg body weight) for a period comprising 7 days before mating, during gestation and until 21 days post-partum. Their brains were then perfusion-fixed and examined for histology and calbindin D-28k protein-immunoreactivity. Histological observations revealed no significant changes in the various brain regions; whereas a reduced number of calbindin-positive cells was encountered in the cingulate and parietal cortices and the lateral septal regions of morphine-treated brains compared with those of controls. The alteration in the expression-patterns of this neuroprotective calcium-binding protein in specific regions of the adult brain might be one of the mechanisms by which the addictive drugs modify the functional aspects of the CNS.

Time dependence of endothelium-mediated vasodilation by intermittent antegrade warm blood cardioplegia.

BACKGROUND: The technique of intermittent antegrade warm blood cardioplegia (IAWBC) exposes the heart to brief periods of normothermic ischemia. This may impair endothelial function in coronary arteries. METHODS: Three cardioplegic technique were tested in porcine hearts arrested for 32 to 36 minutes and reperfused for 30 minutes: IAWBC, antegrade cold blood cardioplegia (ACBC), and antegrade cold crystalloid cardioplegia (ACCC). In the hearts arrested with IAWBC, three different intervals of ischemia were used: three 10-minute intervals (IAWBC1), two 15-minute intervals (IAWBC2), and one 30-minute interval (IAWBC3). Rings from the coronary arteries were used to evaluate in vitro the contractile responses to U46619 and the relaxant responses to bradykinin, A23187, and sodium nitroprusside. RESULTS: All six groups (treatment groups and control group) displayed similar responses to U46619 (30 nmol/L) and nitroprusside. In the IAWBC1, IAWBC2, AND ACBC groups, endothelium-dependent relaxations to bradykinin and A23187 were preserved compared with controls, whereas those of the ACCC and IAWBC3 groups were significantly impaired (bradykinin: control, 8.72 +/- 0.07; IAWBC1, 8.73 +/- 0.03; IAWBC2, 8.65 +/- 0.05; IAWBC3, 8.30 +/- 0.07 [p < 0.05]; ACBC, 8.50 +/- 0.03; ACCC, 8.25 +/- 0.09 [p < 0.05]; A23187: control, 7.07 +/- 0.08; IAWBC1, 7.07 +/- 0.06; IAWBC2, 7.04 +/- 0.03; IAWBC3, 6.64 +/- 0.01 [p < 0.05]; ACBC, 6.80 +/- 0.05; ACCC, 6.60 +/- 0.08 [p < 0.05]; nitroprusside: control, 6.19 +/- 0.1; IAWBC1, 6.19 +/- 0.07; IAWBC2, 6.03 +/- 0.03; IAWBC3, 6.08 +/- 0.05; ACBC, 6.04 +/- 0.2; ACCC, 6.05 +/- 0.03; all values are expressed as the negative logarithm of the concentration producing 50% of the maximal response). CONCLUSIONS: Myocardial preservation with IAWBC with ischemic intervals of 15 minutes or shorter does not alter the endothelium-dependent relaxation to bradykinin or A23187 in porcine coronary arteries, but these responses are significantly impaired by ACCC and IAWBC with an ischemic interval of 30 minutes.

Antifungal and immunoadjuvant properties of fluconazole in mice immunosuppressed with morphine.

We investigated the efficacy of fluconazole on experimental disseminated candidiasis in mice immunocompromised by chronic morphine treatment. CD1 mice were severely immunosuppressed by repeated morphine administrations, i.e., subcutaneous (s.c.) injections of 75 mg/kg/day, 3 days before and 5 days after a systemic Candida albicans infection induced by intravenous administration of 1 x 10(6) fungal cells/mouse. Fluconazole (2.5 mg/kg, s.c., at 6, 24 and 48 h postinfection) was very effective in prolonging survival time of morphine-treated mice. Fluconazole treatment also promotes a recovery of killing activity of polymorphonuclear leukocyte cells suppressed by morphine administrations.

Cocaine increases Sendai virus replication in cultured epithelial cells: critical role of the intracellular redox status.

Cocaine was found to increase parainfluenza-1 Sendai virus (SV) replication in Madin Darby canine kidney (MDCK) cells. Its effect was maximal when it was added before SV infection, while practically no effect was observed when cocaine was added at the time of or after infection. Enhanced SV replication was associated with increased viral protein expression. Cocaine also greatly reduced the intracellular level of glutathione (GSH), namely the most abundant cell thiol with antioxidant functions, recently proposed as an important factor influencing viral infection. Support for this view was provided in the present study by the reversal of cocaine-induced enhancement of SV replication when the intracellular content of GSH was restored by addition of exogenous GSH.

Splenic CD4+ and CD8+ T cells from influenza immune mice concurrently produce in vitro IL2, IL4, and IFN-gamma.

The cytokine responses exerted by virus-primed spleen T cells upon in vitro restimulation were studied. Spleen cells obtained from mice injected intraperitoneally with A/PR8 (H1N1) influenza virus (PR8) were restimulated in vitro with UV-inactivated PR8 virus. The percentage of both CD4+ and CD8+ T cells producing IL2, IL4, or IFN-gamma was assayed at the single cell level by flow cytometric analysis of intracytoplasmic cytokine content. In parallel, the levels of the different cytokines in spleen cell culture supernatants were quantitated by enzyme linked immunosorbent assay. The results showed that in vitro virus restimulation of immune spleen cells induced the concurrent increase, in both CD4+ and CD8+ T cells, of the frequency of IL2-, IFN-gamma-, and IL4-producing cells. The frequency of IFN-gamma-producing T cells was found to be significantly higher in CD8+ T cells. Significant levels of the three cytokines were also detected in the culture supernatants. These data suggest that both CD4+ and CD8+ T cells play an important role in cytokine response to virus infection and that the synthesis and secretion of antiviral and regulatory cytokines is not mutually exclusive either between or within the two T cell subsets. The results of the experiments also indicated that the virus restimulation did not induce a dominant type 1 or type 2 cytokine response.

In vitro effects of cocaine on cytokine secretion induced in murine splenic CD4+ T cells by antigen-specific stimulation.

The in vitro effects of cocaine on antigen-specific-induced cytokine production by murine splenocytes was evaluated both by quantitation by ELISA of the cytokines in culture supernatants and by flow cytometric analysis of the frequency of the cytokine-producing CD4+ T cells. Spleen cells from mice immunized with ovalbumin (OVA) were restimulated with OVA in the presence or absence of cocaine for different periods of time and then evaluated for production of cytokines. Exposure to cocaine was found to reduce the levels in culture supernatants of IL2 and IFN-gamma, whereas IL4 and IL5 levels were not changed. Flow cytometric analysis showed that cocaine increased the frequency of IL2- but not of IL4-producing CD4+ T cells. Kinetics studies indicated that the in vitro antigen-specific-induced production of IL2 is faster than that of IL4 and that cocaine did not affect the production kinetics of either cytokine. Collectively, the results suggest that in vitro cocaine acts by interfering with the secretion rather than with the synthesis of cytokines and that the drug exerts different effects on cytokines with different production kinetics.