The impact of a non-restrictive Antimicrobial Stewardship Program in the emergency department of a secondary-level Italian hospital.

Evidence supporting the effectiveness of Antimicrobial Stewardship (AMS) Programs in the emergency department (ED) setting is limited. We conducted a prospective cohort study to assess the efficacy of an AMS program in an ED and a short-stay observation unit. The intervention included periodic prospective audits (twice a week), conducted by four infectious disease consultants. Primary outcomes included the difference in the hospital mortality rate, antibiotic consumption, and the incidence of bloodstream infections (BSI) caused by multidrug resistant (MDR) bacteria, before March 2020-February 2021 and after March 2021-February 2022 when the program was implemented. Interrupted time-series analysis was performed to assess the effect of our program. During the 12-month program, we performed 152 audits and evaluated 366 antibiotic therapies out of a total of 853 patients admitted. In the intervention period, we observed a non-statistically significant decrease in total antibiotic consumption, with a change in level of - 31.2 defined daily dose/100 patient-days (PD) (p = 0.71). Likewise, we found no significant variations in the rate of BSI due to MDR Gram-positive (CT - 0.02 events/PD, p = 0.84), MDR Gram-negative bacteria (CT 0.08, p = 0.71), or Candida spp. (CT 0.008, p = 0.86). Conversely, we found a significant decrease in the mortality rate between the pre- and post-intervention periods (- 1.98 deaths/100 PD, CI - 3.9 to - 0.007, p = 0.049). The Antibiotic Stewardship Program in the ED was associated with a significant decrease in the mortality rate. More high-quality studies are needed to determine the most effective ASP strategies in this unique setting.

Evidence for IL-6 promoter nuclear activation in U937 cells stimulated with Salmonella enterica serovar Typhimurium porins.

Interleukin-6 (IL-6) is a pleiotropic cytokine and plays an active role in inflammatory and immune responses, contributing to a multitude of physiological and pathophysiological processes. In this study, we address the molecular mechanism of IL-6 transcriptional induction and propose a correlation between activated NF-kappaB localization and IL-6 expression. In particular, we detected, by ChIP assay, that occupation of the IL-6 gene promoter site is dependent on activated NF-kappaB. In fact, after porin stimulation, the NF-kappaB p65 subunit is activated, translocates to the nucleus and binds to the IL-6 promoter sequence.Elucidation of the host signaling pathways and identification of the transcription factors that contribute to IL-6 expression, may aid in the understanding of host susceptibility to gram-negative infections and in identifying new therapeutic strategies in a variety of infectious diseases.

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity.

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Pathophysiological changes of gram-negative bacterial infection can be reproduced by a synthetic peptide mimicking loop L7 sequence of Haemophilus influenzae porin.

Several in vivo models have been used to dissect the molecular mechanisms that contribute to activate the coagulation and fibrinolytic systems by bacteria and bacterial products but many aspects remain poorly understood. In this study we examined the in vivo effect of the synthetic peptide corresponding to loop L7 from Haemophilus influenzae type b (Hib) porin to evaluate its role on the coagulative/fibrinolytic cascade and the circulating markers of endothelial injury. Plasma was obtained from rats injected intravenously with loop L7, Hib porin or a scrambled peptide and tested for fragment 1+2 (F1+2), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type I (PAI-1) antigen, von Willebrand factor (vWF) and soluble E-selectin (sE-selectin). The coagulative/fibrinolytic cascade was impaired as shown by PAI-1 level increased. Concomitantly, E-selectin, a marker of endothelial injury, was also significantly elevated. In addition either loop L7 or Hib porin injection induced hyperglycaemia and inflammatory cytokine production. The data were correlated with hemodynamic functions. The results indicate that loop L7 plays an essential role in the pathophysiologic events observed during gram-negative infection. These findings may have implications for the development of alternative therapies to counteract excessive inflammatory responses during septic shock.

The identification and characterization of fusogenic domains in herpes virus glycoprotein B molecules.

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV-1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post-translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

Role of mitogen-activated protein kinases in the iNOS production and cytokine secretion by Salmonella enterica serovar Typhimurium porins.

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both physiological and pathological functions. The present study examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and proinflammatory cytokine production in RAW 264.7 cells in response to Salmonella enterica serovar Typhimurium porins. By use of Western blotting for iNOS detection and enzyme-linked immunosorbent assay (ELISA) for quantization of cytokine secretion, selective pharmacological inhibitors of MAPK pathways were tested for dissecting the molecular mechanisms underlying the mediation of these signaling in porins-stimulated murine macrophages. S. enterica serovar Typhimurium porins activated iNOS expression, NO production and interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) release. Treatment of cells with SB203580 and SP600125 (inhibitors of p38 and JNK, respectively) significantly affected porin-stimulated iNOS and NO production. Concomitant decrease in the proinflammatory cytokine secretion was detected. These data confirm the importance of the MAPKs cascade in macrophage activation by bacterial product opening up new strategies for therapy of septic shock.

Evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein H in membrane fusion and virus inhibition.

We have identified a putative membrane-interacting domain preceding the transmembrane domain of the Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH). Peptides derived from this region interact strongly with membranes and show a high tendency to partition at the interface. This region is predicted to bind at the membrane interface by adopting an alpha helical structure. Peptides representing either the HSV-1 gH pretransmembrane region or a scrambled control with a different hydrophobic profile at the point of interface have been studied. The peptides derived from this domain of gH induce the fusion of liposomal membranes, adopt helical conformations in membrane mimetic environments and are able to inhibit HSV-1 infectivity. The pretransmembrane region appears to be a common feature in viral fusion proteins of several virus families, and such a feature might be related to their fusogenic function. The identification of membrane-interacting regions capable of modifying the biophysical properties of phospholipid membranes lends weight to the view that such domains might function directly in the fusion process and could facilitate the future development of HSV-1 entry inhibitors.

Structural requirements for proinflammatory activity of porin P2 Loop 7 from Haemophilus influenzae.

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children, characterized by inflammation mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. Synthetic peptides corresponding to loops L5, L6, and L7 activate JNK and p38 mitogen-activated protein kinase (MAPK) pathways, L7 being the most active peptide. Therefore, sequence-activity relationships and key residues were identified by elongating sequence to different extents, designing cyclic peptides, and performing an alanine scan of L7. The ability of mutant peptides to induce activation of signal transduction pathways and release of TNF-alpha and IL-6 has been determined, and, in conjunction with CD spectra, bioinformatics analysis, and molecular dynamics data, showed that 6 out of 8 amino acids contribute significantly to the overall activity. Molecular dynamics showed that L7 modifications increased loop rigidity and helicity after Gly6 mutation, thus, providing a possible structural explanation for observed loss of bioactivity. This work provides insights into essential molecular details of P2 that may impact on the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.

Analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins H and B.

Human herpesviruses enter cells by fusion of their own membrane with a cellular membrane through the concerted action of multiple viral proteins and cellular receptors. Two conserved viral glycoproteins, gB and gH, are required for herpes simplex virus type 1 (HSV-1)-mediated membrane fusion, but little is known of how these proteins cooperate during entry. Both glycoproteins were shown to contain heptad repeat (HR) sequences predicted to form alpha-helical coiled coils, and the inhibitory activity against infection of four sets of synthetic peptides corresponding to HR1 and HR2 of gB and gH was tested. The interactions between these HR peptides were also investigated by circular dichroism, native polyacrylamide-gel electrophoresis and size exclusion high-performance liquid chromatography. gH coiled-coil peptides were more effective than gB coiled-coils peptides in inhibiting virus infectivity. The peptides did not impair fusion when added to cells immediately after infection. In contrast, inhibition of infection was observed, albeit to various extents, when peptides were added to virus before or during inoculation. The results of biophysical analyses were indicative of the existence of an interaction between HR1 and HR2 of gH and suggest that the HRs of gB and gH do not interact with each other.

STAT1 and STAT3 phosphorylation by porins are independent of JAKs but are dependent on MAPK pathway and plays a role in U937 cells production of interleukin-6.

A group of transcription factors, termed signal transducers and activators of transcription (STATs), appears to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Similarly, cytoplasmic Janus kinases (JAKs) seem to play a critical role in diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. In this work, we analysed the effects of the Salmonella enterica serovar Typhimurium porins on signaling by the JAK/STAT pathway and IL-6 release in U937 cells. Porins and LPS of membrane from Gram-negative bacteria are factors implicated in septic shock. In our assays porins induce interleukin-6 (IL-6) release (110+/-2.6pg/ml) 24h after stimulation and STAT1/STAT3 tyrosine (Tyr701/Tyr705) and serine (Ser727) phosphorylation after 15min. By using several selective inhibitors we demonstrate that porins modulate the activation of STAT1/STAT3 through mitogen activated protein kinases (MAPKs) and not JAKs. Furthermore, we demonstrated that STAT1 and STAT3 are not involved in the modulation of IL-6 release in U937 cells stimulated with porins. Inhibition of tyrosine/serine phosphorylation mediated by MAPKs of STAT1 and STAT3 decrease the IL-6 secretion following porin stimulation. Therefore, suggesting a key role of this pathway in phosphorylation of Ser 727 in STAT1 and STAT3. These results are confirmed by porin or LPS-induced nuclear translocation of STAT1 and STAT3 in U937 cells.

Heat shock protein 27 expression in patients with chronic liver damage.

The aim of this study was to evaluate a possible relationship between lymphomonocyte expression of heat shock proteins (HSP) 60/27 and plasma levels of pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and markers of antioxidant/oxidative status [glutathione (GSH), alpha glutathione-S-transferase activity (alpha GST), malonyldialdeyde (MDA), 4-hydroxinonenal (4-HNE), and S-nitrosothiols (S-NO)] in patients with chronic liver diseases. Entered into the study were 47 subjects: 10 healthy controls, 16 patients with HCV-related chronic hepatitis (CH), and 16 patients with HCV-related and 5 with alcohol-related liver cirrhosis (10 Child A and 11 Child B+C). HSP60 was clearly expressed only in 5% of patients and lowly in the control group. HSP27 was clearly expressed in 46.7% of CH and 71.4% of cirrhotic patients but was lowly present in healthy subjects. A significant difference was found between patients with a low expression of HSP27 (negative patients) and those with a high HSP27 expression (positive patients) of plasma levels both of antioxidants (GSH, p < 0.05), and of markers of enhanced production of free radicals and cytokines (alpha GST, TNF-alpha and IL-6, p < 0.05; MDA, 4-HNE and S-NO, p < 0.01) as well as for alcohol use and degree of liver impairment. The present data are the first showing that, particularly in conditions of enhanced oxidative stress, lymphomonocytes from liver disease patients present an increased expression of HSP27.

Prolactin modulates IL-8 production induced by porins or LPS through different signaling mechanisms.

Prolactin (PRL) induces cell proliferation and cell differentiation through the well-known mitogen-activated protein kinases (MAPKs) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, depending on the cell line. MAPKs play a central role in signaling transduction mechanisms that transmit mitogenic or differentiation signals from an activated receptor to the intracellular machinery. All of the cytokine receptors that activate the JAK/STAT pathway also activate the MAPK pathway. The aim of the present study was to delineate the signal pathways implicated in IL-8 release by THP-1 cells, pretreated with PRL, after stimulation with either lipopolysaccharide (LPS) or porins from Salmonella enterica serovar Typhimurium. PRL activates the JAK2/STAT1-3 signaling pathway, while LPS or porins from S. enterica serovar Typhimurium does not induce any phosphorylation of this pathway. However, in THP-1 cells, the combination of PRL followed by either S. enterica serovar Typhimurium LPS or porins produced a greater MEK1-MEK2/MAPKs activation response than treatment with PRL alone. Similarly, PRL pretreatment of THP-1 cells resulted in an increase in IL-8 release in response to stimulation with either LPS or porins. This additive effect on IL-8 release was reduced when the cells were also treated with PD-098059, a selective inhibitor of the MEK1 activator and the MAPK cascade, or SB203580, a specific inhibitor of the p38 pathway, or AG490, a specific JAK/STAT pathway inhibitor, providing evidence that there are different signal pathways activated which have a cumulative effect.

Induction of signaling pathways by herpes simplex virus type 1 through glycoprotein H peptides.

Eukaryotic cells respond to extracellular stimuli, such as viruses, by recruiting signal transduction pathways, many of which are mediated through activation of distinct mitogen-activated protein kinase (MAPK) cascades and activation of transductional regulation factors. The best characterized of this pathway are the extracellular signal regulated kinase (ERK), the c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), and the p38 MAPK cascade. Herpes simplex virus type 1 (HSV-1) encodes at least 11 envelope glycoproteins, which alone or in concert play different roles in viral adsorption, entry, cell-to-cell spread, and immune evasion. Of these proteins, three are designated glycoprotein B (gB), glycoprotein D (gD), and the gH/gL heterodimer, are clearly involved in attachment and entry, and therefore possible candidates in inducing early cellular activation.Nevertheless, the precise role of each glycoprotein and the cellular factor involved remain elusive. The signal transduction pathways involved, and the outcome of cellular activation on viral entry or postentry events, are still to be elucidated. To better understand the role of signal transduction pathways and phosphorylation events in HSV-1 entry, synthetic peptides modeled on HSV-1 gH were synthesized and tested for MEK1-MEK2/MAPK cascade activation. Our results show a major involvement of the JNK pathway in the intracellular signal transmission after stimulation with gH HSV-1 peptides.

Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade.

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the beta-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal beta strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.

Porins from Salmonella enterica serovar Typhimurium activate the transcription factors activating protein 1 and NF-kappaB through the Raf-1-mitogen-activated protein kinase cascade.

In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-kappaB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2'-amino-3'-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-kappaB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-kappaB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-kappaB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-kappaB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS.

Porins from Salmonella enterica serovar Typhimurium induce TNF-alpha, IL-6 and IL-8 release by CD14-independent and CD11a/CD18-dependent mechanisms.

Lipopolysaccharide (LPS) of Gram-negative bacteria and several surface components of Gram-positive bacteria utilize CD14 and CD11a/18 as cellular receptors to induce expression and release of cytokines. Of the surface components of Gram-negative bacteria, porins exhibit a biological activity similar to that of LPS. The results in this paper show that the mechanism of stimulation by porins of THP-1 cells enriched in CD14 receptor after treatment with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) is independent of this receptor, but is partially dependent on CD11a/18 integrins.