Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

The magnitude of septic shock as a clinical problem is often understated. Despite advances in our ability to diagnose and treat infectious diseases, severe sepsis leading to shock due to gram-negative infections remains one of the leading causes of mortality worldwide. Septic shock develops because of a disregulation in the host response, and the mechanisms initially recruited to fight infection produce life-threatening tissue damage and death. Recent research has witnessed a significant increase in our understanding of host-pathogen interactions, particularly in the area of innate immunity and the molecular recognition of gram-positive and gram-negative bacteria. Important new mediators of sepsis and novel mechanisms of host-cell toxicity have been identified and, together with clinical trials targeting pathways considered central to sepsis pathogenesis, provide new insight into the molecular and cellular basis of sepsis for the formulation of new strategies of intervention. Research on septic shock pathogenesis by gram-negative bacteria is mainly focused on the understanding of the molecular and cellular role played by lipopolysaccharide (LPS). Strong experimental evidence and clinical observations suggest that the release of proinflammatory cytokine mediators by LPS-responsive cells (mainly macrophages, endothelial cells and neutrophils) in response to toxic products sets in motion the genetic and physiologic program that manifests as shock. The best characterized of these toxic components is LPS, which is considered as a paradigm for other less well-characterized toxic microbial molecules. The immune protection stimulated by highly purified LPS in animals does not resolve the symptomatology of septic shock, while LPS mixed to outer membrane proteins shows a better protective activity. Several studies evidence the major role played by outer membrane proteins in the molecular interaction between the host cell and the gram-negative bacteria. Endotoxin-associated proteins consist of a complex of several major proteins that are intimately associated with the LPS. Very little is known about release of non-LPS gramnegative outer membrane components such as OMPs in sepsis. Among the OMPs, porins have been shown to play an important role in pathogenesis of bacterial infections. Porins were pyrogenic in rabbits and elicited a localized reaction when used as the sensiting and eliciting agent. Porins were also shown to kill D-galactosamine sensitized LPS-responsive and LPS-unresponsive mice. Treatment of Human Umbilical Vein Endothelial Cells: (HUVEC) with porins increased the transmigration of different leukocyte populations, inparticular of neutrophils. Porins by several gram-negative bacteria induce cytokine release by human leukocytes as well as enhancement of cytokine gene expression. Also, other components of the bacterial envelope are important in the induction and pathogenesis of septic shock such as bacterial lipoproteins (LP). As anti-LPS therapies does not seem to improve by the addition of proteins from the outer membrane or small fragments of these proteins, a great alternative to existing strategies will involve the blockage of signal transduction pathways, cytokine and inflammatory mechanisms

Review of a viral peptide nanosystem for intracellular delivery

The internalization of bioactive molecules is one of the most critical problems to overcome in theranostics. In order to improve pharmacokinetic and pharmacodynamic proper- ties, synthetic transporters are widely investigated. A new nanotechnological transporter, gH625, is based on a viral peptide sequence derived from the herpes simplex virus type 1 glycoprotein H (gH) that has proved to be a useful delivery vehicle, due to its intrinsic properties of inducing membrane perturbation. The peptide functionalization with several kinds of nanoparticles like quantum dots, dendrimers, and liposomes could be of particular interest in biomedical applica- tions to improve drug release within cells, to increase site-specific action, and eventually to reduce related cytotoxicity. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. (DOI: 10.1117/1.JNP.7.071599

Biophysical Characterization and Membrane Interaction of the Two Fusion Loops of Glycoprotein B from Herpes Simplex Type I Virus

The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV) requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p < 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p < 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p < 0.0001) or urgent (20.4% vs. 38.5%; p < 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p < 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

NEW BIOTECHNOLOGICAL METHODS FOR INTRACELLULAR DELIVERY

Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target. In order to overcome this barrier-like behavior, not easily circumvented by many pharmacologically-active molecules, and to promote cellular uptake, synthetic peptide-based transporters have been exploited. In fact, linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of Cell Penetrating Peptides (CPPs) is one of the most promising non invasive strategies to efficiently translocate macromolecules through the plasma membrane, and have, recently, attracted a great attention. A new viral traslocator peptide (gH625) is described in this thesis, which is derived from the Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH). gH625 has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation. Quantum dots (QDs), Liposomes, Dendrimers and Polystyrene Nanoparticles (NPs) have been used as powerful platforms for studying the behavior of gH625 as a nano-vehicles. QDs are a new class of fluorescent probes under intense research and development for broad application in molecular, cellular and in vivo imaging. QDs do not significantly traverse the membrane bilayer on their own. On the contrary, when functionalized with gH625, are able to translocate through cellular plasma membranes by a mechanism which seems to be only relatively dependent on the endocytic route of entry. Liposomal aggregates have been successfully used as in vivo carriers of active principles; they display some unique pharmacokinetic properties and can be adapted to a wide range of therapeutic agents. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of gH625 peptide on the external surface of DOPG based liposomes. Liposomes have been also loaded with the cytotoxic doxorubicin drug and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells. Dendrimers are perfectly hyperbranched macromolecules with a well-defined structure, which exhibit properties very different from linear polymers with the same composition and molecular weight. A poly(amide)-based dendrimer was synthesized and conjugated with gH625. The attachment of the peptide sequences to the termini of a dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. The peptide functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism. The surface of fluorescent aminated polystyrene nanoparticles (NPs) was functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro Blood Brain Barrier (BBB) models were studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances their BBB crossing. In summary, these results establish that gH625 may represent a good choice for the design of promising carriers to deliver drugs for the treatment of human diseases

Generation effect of Newkome dendrimers on cellular uptake

Poly(amide)-based dendrimers can be used as delivery scaffolds in conjunction with the cell-penetrating peptide gH625 derived from the glycoprotein of the Herpes Simplex virus type 1. In this contribution, we aim to isolate the optimal dendrimer generation for cellular uptake for Newkome type dendrimers conjugated with gH625. For this study, we synthesized generations zero to three of the Newkome dendrimer-gH625 bioconjugate. Fluorescent microscopy experiments showed that the second and third generations are the most efficient for cellular uptake with the second generation having the synthetic advantage. The optimal second generation can be used as an improved material for a dendrimer based delivery scaffold for peptide therapeutics

Elucidation of the interaction mechanism with liposomes of gH625-peptide functionalized dendrimers

We have demonstrated that amide-based dendrimers functionalized with the membrane-interacting peptide gH625 derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H enter cells mainly through a non-active translocation mechanism. Herein, we investigate the interaction between the peptide-functionalized dendrimer and liposomes composed of PC/Chol using fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance to get insights into the mechanism of internalization. The affinity for the membrane bilayer is very high and the interaction between the peptide-dendrimer and liposomes took place without evidence of pore formation. These results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery

A viral peptide for intracellular delivery

Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

Peptides complementary to the active loop of porin P2 from Haemophilus influenzae modulate its activity.

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children. It is characterized by inflammation that is 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. A synthetic peptide corresponding to loop L7 of the porin activates JNK and p38 mitogenactivated protein kinase (MAPK) pathways. We report a novel use of the complementary peptide approach to design a peptide that is able to bind selectively to the protein P2, thereby reducing its activity. This work provides insights into essential molecular details of P2 that may affect the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy. © 2012 Cantisani et al, publisher and licensee Dove Medical Press Ltd