Low Percentage of Perforin-Expressing NK Cells during Severe SARS-CoV-2 Infection: Consumption Rather than Primary Deficiency.

Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.

Post-acute sequelae of COVID-19 is characterized by diminished peripheral CD8+ß7 integrin+ T cells and anti-SARS-CoV-2 IgA response.

Several millions of individuals are estimated to develop post-acute sequelae SARS-CoV-2 condition (PASC) that persists for months after infection. Here we evaluate the immune response in convalescent individuals with PASC compared to convalescent asymptomatic and uninfected participants, six months following their COVID-19 diagnosis. Both convalescent asymptomatic and PASC cases are characterised by higher CD8+ T cell percentages, however, the proportion of blood CD8+ T cells expressing the mucosal homing receptor ß7 is low in PASC patients. CD8 T cells show increased expression of PD-1, perforin and granzyme B in PASC, and the plasma levels of type I and type III (mucosal) interferons are elevated. The humoral response is characterized by higher levels of IgA against the N and S viral proteins, particularly in those individuals who had severe acute disease. Our results also show that consistently elevated levels of IL-6, IL-8/CXCL8 and IP-10/CXCL10 during acute disease increase the risk to develop PASC. In summary, our study indicates that PASC is defined by persisting immunological dysfunction as late as six months following SARS-CoV-2 infection, including alterations in mucosal immune parameters, redistribution of mucosal CD8+ß7Integrin+ T cells and IgA, indicative of potential viral persistence and mucosal involvement in the etiopathology of PASC.

T4 apoptosis in the acute phase of SARS-CoV-2 infection predicts long COVID.

Background: As about 10% of patients with COVID-19 present sequelae, it is important to better understand the physiopathology of so-called long COVID. Method: To this aim, we recruited 29 patients hospitalized for SARS-CoV-2 infection and, by Luminex®, quantified 19 soluble factors in their plasma and in the supernatant of their peripheral blood mononuclear cells, including inflammatory and anti-inflammatory cytokines and chemokines, Th1/Th2/Th17 cytokines, and endothelium activation markers. We also measured their T4, T8 and NK differentiation, activation, exhaustion and senescence, T cell apoptosis, and monocyte subpopulations by flow cytometry. We compared these markers between participants who developed long COVID or not one year later. Results: None of these markers was predictive for sequelae, except programmed T4 cell death. T4 lymphocytes from participants who later presented long COVID were more apoptotic in culture than those of sequelae-free participants at Month 12 (36.9 ± 14.7 vs. 24.2 ± 9.0%, p = 0.016). Conclusions: Our observation raises the hypothesis that T4 cell death during the acute phase of SARS-CoV-2 infection might pave the way for long COVID. Mechanistically, T4 lymphopenia might favor phenomena that could cause sequelae, including SARS-CoV-2 persistence, reactivation of other viruses, autoimmunity and immune dysregulation. In this scenario, inhibiting T cell apoptosis, for instance, by caspase inhibitors, could prevent long COVID.

Low perforin expression in CD8+ T lymphocytes during the acute phase of severe SARS-CoV-2 infection predicts long COVID.

T cell cytotoxicity plays a major role in antiviral immunity. Anti-SARS-CoV-2 immunity may determine acute disease severity, but also the potential persistence of symptoms (long COVID). We therefore measured the expression of perforin, a cytotoxic mediator, in T cells of patients recently hospitalized for SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to Intensive Care Units (ICUs) or non-ICU, and 29 age- and sex-matched healthy controls (HCs). Amounts of intracellular perforin and granzyme-B, as well as cell surface expression of the degranulation marker CD107A were determined by flow cytometry. The levels of 15 cytokines in plasma were measured by Luminex. The frequency of perforin-positive T4 cells and T8 cells was higher in patients than in HCs (9.9 ± 10.1% versus 4.6 ± 6.4%, p = 0.006 and 46.7 ± 20.6% vs 33.3 ± 18.8%, p = 0.004, respectively). Perforin expression was neither correlated with clinical and biological markers of disease severity nor predictive of death. By contrast, the percentage of perforin-positive T8 cells in the acute phase of the disease predicted the onset of long COVID one year later. A low T8 cytotoxicity in the first days of SARS-CoV-2 infection might favor virus replication and persistence, autoimmunity, and/or reactivation of other viruses such as Epstein-Barr virus or cytomegalovirus, paving the way for long COVID. Under this hypothesis, boosting T cell cytotoxicity during the acute phase of the infection could prevent delayed sequelae.

Low quantity and quality of anti-spike humoral response is linked to CD4 T-cell apoptosis in COVID-19 patients.

In addition to an inflammatory reaction, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-infected patients present lymphopenia, which we recently reported as being related to abnormal programmed cell death. As an efficient humoral response requires CD4 T-cell help, we hypothesized that the propensity of CD4 T cells to die may impact the quantity and quality of the humoral response in acutely infected individuals. In addition to specific immunoglobulins (Ig)A, IgM, and IgG against SARS-CoV-2 nucleocapsid (N), membrane (M), and spike (S1) proteins, we assessed the quality of IgG response by measuring the avidity index. Because the S protein represents the main target for neutralization and antibody-dependent cellular cytotoxicity responses, we also analyzed anti-S-specific IgG using S-transfected cells (S-Flow). Our results demonstrated that most COVID-19 patients have a predominant IgA anti-N humoral response during the early phase of infection. This specific humoral response preceded the anti-S1 in time and magnitude. The avidity index of anti-S1 IgG was low in acutely infected individuals compared to convalescent patients. We showed that the percentage of apoptotic CD4 T cells is inversely correlated with the levels of specific IgG antibodies. These lower levels were also correlated positively with plasma levels of CXCL10, a marker of disease severity, and soluble Fas ligand that contributes to T-cell death. Finally, we found lower S-Flow responses in patients with higher CD4 T-cell apoptosis. Altogether, these results demonstrate that individuals with high levels of CD4 T-cell apoptosis and CXCL10 have a poor ability to build an efficient anti-S response. Consequently, preventing CD4 T-cell death might be a strategy for improving humoral response during the acute phase, thereby reducing COVID-19 pathogenicity.

Angiotensin II induces reactive oxygen species, DNA damage, and T-cell apoptosis in severe COVID-19.

BACKGROUND: Lymphopenia is predictive of survival in patients with coronavirus disease 2019 (COVID-19). OBJECTIVE: The aim of this study was to understand the cause of the lymphocyte count drop in severe forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Monocytic production of reactive oxygen species (ROSs) and T-cell apoptosis were measured by flow cytometry, DNA damage in PBMCs was measured by immunofluorescence, and angiotensin II (AngII) was measured by ELISA in patients infected with SARS-CoV-2 at admission to an intensive care unit (ICU) (n = 29) or not admitted to an ICU (n = 29) and in age- and sex-matched healthy controls. RESULTS: We showed that the monocytes of certain patients with COVID-19 spontaneously released ROSs able to induce DNA damage and apoptosis in neighboring cells. Of note, high ROS production was predictive of death in ICU patients. Accordingly, in most patients, we observed the presence of DNA damage in up to 50% of their PBMCs and T-cell apoptosis. Moreover, the intensity of this DNA damage was linked to lymphopenia. SARS-CoV-2 is known to induce the internalization of its receptor, angiotensin-converting enzyme 2, which is a protease capable of catabolizing AngII. Accordingly, in certain patients with COVID-19 we observed high plasma levels of AngII. When looking for the stimulus responsible for their monocytic ROS production, we revealed that AngII triggers ROS production by monocytes via angiotensin receptor I. ROSs released by AngII-activated monocytes induced DNA damage and apoptosis in neighboring lymphocytes. CONCLUSION: We conclude that T-cell apoptosis provoked via DNA damage due to the release of monocytic ROSs could play a major role in COVID-19 pathogenesis.

Leishmania infantum Infection of Primary Human Myeloid Cells.

Circulating phagocytic cells often serve as cellular targets for a large number of pathogens such as Leishmania parasites. Studying primary human cells in an infectious context requires lengthy procedures for cell isolation that may affect the analysis performed. Using whole blood and a no-lyse and no-wash flow cytometric assay (NoNo assay), we monitored the Leishmania infantum infection of primary human cells. We demonstrated, using fluorescent parasites, that among monocyte cell populations, L. infantum preferentially infects classical (CD14+CD16-) and intermediate (CD14+CD16+) primary human monocytes in whole blood. Because classical monocytes are the preponderant population, they represent the larger L. infantum reservoir. Moreover, we also found that, concomitantly to monocyte infection, a subset of PMNs is infected early in whole blood. Of interest, in whole blood, PMNs are less infected compared to classical monocytes. Overall, by using this NoNo assay, we provided a novel avenue in our understanding of host-leishmania interactions.

T cell apoptosis characterizes severe Covid-19 disease.

Severe SARS-CoV-2 infections are characterized by lymphopenia, but the mechanisms involved are still elusive. Based on our knowledge of HIV pathophysiology, we hypothesized that SARS-CoV-2 infection-mediated lymphopenia could also be related to T cell apoptosis. By comparing intensive care unit (ICU) and non-ICU COVID-19 patients with age-matched healthy donors, we found a strong positive correlation between plasma levels of soluble FasL (sFasL) and T cell surface expression of Fas/CD95 with the propensity of T cells to die and CD4 T cell counts. Plasma levels of sFasL and T cell death are correlated with CXCL10 which is part of the signature of 4 biomarkers of disease severity (ROC, 0.98). We also found that members of the Bcl-2 family had modulated in the T cells of COVID-19 patients. More importantly, we demonstrated that the pan-caspase inhibitor, Q-VD, prevents T cell death by apoptosis and enhances Th1 transcripts. Altogether, our results are compatible with a model in which T-cell apoptosis accounts for T lymphopenia in individuals with severe COVID-19. Therefore, a strategy aimed at blocking caspase activation could be beneficial for preventing immunodeficiency in COVID-19 patients.

Cytokines and metabolic regulation: A framework of bidirectional influences affecting Leishmania infection.

Leishmania, a protozoan parasite inflicting the complex of diseases called Leishmaniases, resides and replicates as amastigotes within mammalian macrophages. As macrophages are metabolically highly active and can generate free radicals that can destroy this parasite, Leishmania also devise strategies to modulate the host cell metabolism. However, the metabolic changes can also be influenced by the anti-leishmanial immune response mediated by cytokines. This bidirectional, dynamic and complex metabolic coupling established between Leishmania and its host is the result of a long co-evolutionary process. Due to the continuous alterations imposed by the host microenvironment, such metabolic coupling continues to be dynamically regulated. The constant pursuit and competition for nutrients in the host-Leishmania duet alter the host metabolic pathways with major consequences for its nutritional reserves, eventually affecting the phenotype and functionality of the host cell. Altered phenotype and functions of macrophages are particularly relevant to immune cells, as perturbed metabolic fluxes can crucially affect the activation, differentiation, and functions of host immune cells. All these changes can deterministically direct the outcome of an infection. Cytokines and metabolic fluxes can bidirectionally influence each other through molecular sensors and regulators to dictate the final infection outcome. Our studies along with those from others have now identified the metabolic nodes that can be targeted for therapy.

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus).

Amphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agents.

Antileishmanial Drugs Modulate IL-12 Expression and Inflammasome Activation in Primary Human Cells.

Leishmaniases are neglected tropical diseases. The treatment of leishmaniasis relies exclusively on chemotherapy including amphotericin B (AmB), miltefosine (hexadecylphosphocholine), and pentamidine. Besides the fact that these molecules are harmful for patients, little is known about the impact of such antileishmanial drugs on primary human cells in relation to immune function. The present study demonstrates that all antileishmanial drugs inhibit CD4 and CD8 T cell proliferation at the doses that are not related to increased cell death. Our results highlight that antileishmanial drugs have an impact on monocytes by altering the expression of IL-12 induced by LPS, whereas only AmB induced IL-10 secretion; both cytokines are essential in regulating Th1 cell-mediated immunity. Interestingly, IL-12 and anti-IL-10 Abs improved T cell proliferation inhibited by AmB. Furthermore, our results show that in contrast to hexadecylphosphocholine and pentamidine, AmB induced gene expression of the inflammasome pathway. Thus, AmB induced IL-1ß and IL-18 secretions, which are reduced by specific inhibitors of caspase activation (Q-VD) and NLRP3 activation (MCC950). Our results reveal previously underestimated effects of antileishmanial drugs on primary human cells.

In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts.

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.

Non-human primates and Leishmania immunity.

In the context of infectious diseases, non-human primates (NHP) provide the best animal models of human diseases due to the close phylogenetic relationship and the similar physiology and anatomical systems. Herein, we summarized the contribution of NHP models for understanding the immunity to leishmaniases, which are a group of diseases caused by infection with protozoan parasites of the genus Leishmania and classified as one of the neglected tropical diseases.

Transcriptional Analysis of Human Skin Lesions Identifies Tryptophan-2,3-Deoxygenase as a Restriction Factor for Cutaneous Leishmania.

Disease manifestation after infection with cutaneous Leishmania species is the result of a complex interplay of diverse factors, including the immune status of the host, the infecting parasite species, or the parasite load at the lesion site. Understanding how these factors impact on the pathology of cutaneous leishmaniasis (CL) may provide new targets to manage the infection and improve clinical outcome. We quantified the relative expression of 170 genes involved in a diverse range of biological processes, in the skin biopsies from patients afflicted with CL caused by infection with either L. major or L. tropica. As compared to healthy skin, CL lesions bear elevated levels of transcripts involved in the immune response, and conversely, present a significant downregulation in the expression of genes involved in epidermal integrity and arginine or fatty acid metabolism. The expression of transcripts encoding for cytotoxic mediators and chemokines in lesions was inversely correlated with the expression of genes involved in epidermal integrity, suggesting that cytotoxicity is a major mediator of CL pathology. When comparing the transcriptional profiles of lesions caused by either L. major or L. tropica, we found them to be very similar, the later presenting an aggravated inflammatory/cytotoxic profile. Finally, we identified genes positively correlated with the parasite load in lesions. Among others, these included Th2 or regulatory cytokines, such as IL4 or IL10. Remarkably, a single gene among our dataset, encoding for tryptophan-2,3-deoxygenase (TDO), presented a negative correlation with the parasite load, suggesting that its expression may restrict parasite numbers in lesions. In agreement, treatment of macrophages infected with L. major in vitro with a TDO inhibitor led to an increase in parasite transcripts. Our work provides new insights into the factors that impact CL pathology and identifies TDO as a restriction factor for cutaneous Leishmania.

Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent.

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

Structure-Activity Relationship-based Optimization of Small Temporin-SHf Analogs with Potent Antibacterial Activity.

Short antimicrobial peptides represent attractive compounds for the development of new antibiotic agents. Previously, we identified an ultrashort hydrophobic and phenylalanine-rich peptide, called temporin-SHf, representing the smallest natural amphibian antimicrobial peptide known to date. Here, we report on the first structure-activity relationship study of this peptide. A series of temporin-SHf derivatives containing insertion of a basic arginine residue as well as residues containing neutral hydrophilic (serine and α-hydroxymethylserine) and hydrophobic (α-methyl phenylalanine and p-(t)butyl phenylalanine) groups were designed to improve the antimicrobial activity, and their α-helical structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. Three compounds were found to display higher antimicrobial activity with the ability to disrupt (permeabilization/depolarization) the bacterial membrane while retaining the nontoxic character of the parent peptide toward rat erythrocytes and human cells (THP-1 derived macrophages and HEK-293). Antimicrobial assays were carried out to explore the influence of serum and physiological salt concentration on peptide activity. Analogs containing d-amino acid residues were also tested. Our study revealed that [p-(t)BuF(2), R(5)]SHf is an attractive ultrashort candidate that is highly potent (bactericidal) against Gram-positive bacteria (including multidrug resistant S. aureus) and against a wider range of clinically interesting Gram-negative bacteria than temporin-SHf, and also active at physiological salt concentrations and in 30% serum.

Structure, antimicrobial activities and mode of interaction with membranes of novel [corrected] phylloseptins from the painted-belly leaf frog, Phyllomedusa sauvagii.

Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6) and also of phylloseptin-1 (PSN-1, here renamed PLS-S1), the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79-95% amino acid sequence identity; net charge  = +2) were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge  = +1) and -S5 (net charge  = +2) were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of phylloseptins.

Tacrine and its analogues impair mitochondrial function and bioenergetics: a lipidomic analysis in rat brain.

Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer in the treatment of Alzheimer's disease (AD). However, its low therapeutic efficiency and a high incidence of side effects have limited its clinical use. In this study, the molecular mechanisms underlying the impact on brain activity of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing on three aspects: (i) their effects on brain cholinesterase activity; (ii) perturbations on electron transport chain enzymes activities of non-synaptic brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced by these compounds on mitochondrial bioenergetics. Brain effects were evaluated 18 h after the administration of a single dose (75.6 µmol/kg) of tacrine or tacrine analogues. The three compounds promoted a significant reduction in brain AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and less active than T1 tacrine analogue, whereas BuChE inhibition followed the order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show that all the compounds studied disturbed brain mitochondrial bioenergetics mainly via the inhibition of complex I activity. Furthermore, the activity of complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is only affected by tacrine. Therefore, the compounds' toxicity as regards brain mitochondria, which follows the order: tacrine >> T1 > T2, does not correlate with their ability to inhibit brain cholinesterase enzymes. Lipidomics approaches show that phosphatidylethanolamine (PE) is the most abundant phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL) present the greatest diversity of molecular species. Tacrine induced significant perturbations in the mitochondrial PL profile, which were detected by means of changes in the relative abundance of phosphatidylcholine (PC), PE, phosphatidylinositol (PI) and CL and by the presence of oxidized phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid content and molecular composition of brain mitochondria PL are perturbed to a lesser extent than in the tacrine group. Abnormalities in CL content and the amount of oxidized phosphatidylserines were associated with significant reductions in mitochondrial enzymes activities, mainly complex I. These results indicate that tacrine and its analogues impair mitochondrial function and bioenergetics, thus compromising the activity of brain cells.

[Epithelioid hemangioendothelioma of the pleura - A rare presentation of a clinical case]. / Hemangioendotelioma epitelióide da pleura - Uma apresentação rara a propósito de um caso clínico.

Epithelioid Hemangioendothelioma (EHE) is a vascular tumour with rare pleural presentation. As there are a small number of cases described in the literature the authors present the case of a 65 -year -old woman, who was admitted to the Emergency Department for a right -sided chest pain, which progressed over 7 months, after a thoracic trauma. The chest X -ray showed signs of a moderate right -sided pleural effusion. A pleural -pulmonary biopsy carried out by toracotomy established the histological diagnosis of EHE of the pleura. Due to the locally advanced stage of the tumour, chemotherapy with carboplatin and etoposide was prescribed and the patient died 6 months later. This case confirms that pleural EHE has an aggressive behaviour, similar to an angiossarcoma, with a median survival of only a few months after diagnosis.

[Lymphangioleiomyomatosis - report of three cases]. / Linfangioleiomiomatose - A propósito de três casos clínicos.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease of unknown aetiology. It is characterized by proliferation of abnormal smooth -muscle cells throughout the peribronchial, perivascular and perilymphatic regions of the lung. LAM may occur sporadically, in association with tuberous sclerosis complex (TSC) or inheritable multiorgan hamartomatosis. In either situation, LAM occurs almost exclusively in women of reproductive age, and approximately one third of the patients with TSC have LAM2. The authors review the cases of three female patients diagnosed with LAM based on clinical and radiological findings. A brief review of the disease is then presented.