Response to the comment on “Promising blood-derived biomarkers for estimation of the postmortem interval’’

Accepted ManuscriptFollowing Meurs and Szykula's comment on our published article titled "Promising blood-derived biomarkers for estimation of the postmortem interval", we recognize the importance of the issues raised, but we would like to emphasize that these contain some misinterpretations and that most of the points were already discussed in depth in our manuscript particularly in the conclusion section. We also aim to highlight further data regarding the difficulties of postmortem interval estimation

Exploring NAD(+) metabolism in host-pathogen interactions

Nicotinamide adenine dinucleotide (NAD+) is a vital molecule found in all living cells. NAD+ intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD+ metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD+ metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD+ homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD+ or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD+ metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.JG was supported by PD/BD/106053/2015. BV was supported by IRD (Institut de Recherche pour le Développement) institutional funding. JE was supported by a European Community’s Seventh Framework Program under grant agreement No. 602773 (Project KINDRED), an ANR grant (LEISH-APO, France) and a Partenariat Hubert Curien (PHC) (program Volubilis, MA/11/262). JE also thanks the Canada Research Chair program for his support. RS thank FCT—Foundation for Science and Technology—for their Investigator FCT Grant (IF/00021/2014)info:eu-repo/semantics/publishedVersio

Immune tumor microenvironment in ovarian cancer ascites

Ovarian cancer (OC) has a specific type of metastasis, via transcoelomic, and most of the patients are diagnosed at advanced stages with multiple tumors spread within the peritoneal cavity. The role of Malignant Ascites (MA) is to serve as a transporter of tumor cells from the primary location to the peritoneal wall or to the surface of the peritoneal organs. MA comprise cellular components with tumor and non-tumor cells and acellular components, creating a unique microenvironment capable of modifying the tumor behavior. These microenvironment factors influence tumor cell proliferation, progression, chemoresistance, and immune evasion, suggesting that MA play an active role in OC progression. Tumor cells induce a complex immune suppression that neutralizes antitumor immunity, leading to disease progression and treatment failure, provoking a tumor-promoting environment. In this review, we will focus on the High-Grade Serous Carcinoma (HGSC) microenvironment with special attention to the tumor microenvironment immunology.This work was supported by Fundacão para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior and European Union through a PhD fellowship (2021.05081.BD) cosponsored by Fundo Social Europeu (FSE) through Programa Operacional Regional Norte (Norte 2020)

Promising blood-derived biomarkers for estimation of the postmortem interval

A precise estimation of the postmortem interval (PMI) is one of the most important topics in forensic pathology. However, the PMI estimation is based mainly on the visual observation of cadaverous pheno- mena (e.g. algor, livor and rigor mortis) and on alternative methods such as thanatochemistry that remain relatively imprecise. The aim of this in vitro study was to evaluate the kinetic alterations of several bio- chemical parameters (i.e. proteins, enzymes, substrates, electrolytes and lipids) during putrefaction of human blood. For this purpose, we performed kinetic biochemical analysis during a 264 hour period. The results showed a significant linear correlation between total and direct bilirubin, urea, uric acid, transferrin, immunoglobulin M (IgM), creatine kinase (CK), aspartate transaminase (AST), calcium and iron with the time of blood putrefaction. These parameters allowed us to develop two mathematical models that may have predictive values and become important complementary tools of traditional methods to achieve a more accurate PMI estimationFundação para a Ciência e a Tecnologia (FCT) for his Investigator Grant (IF/01147/2013

Evaluating the Role of Host AMPK in Leishmania Burden

Uncorrected proofThe study of host AMP-activated protein kinase (AMPK) activation during Leishmania infection imposes distinct types of techniques to measure protein expression and activation, as well as to quantify, at transcription and translational levels, its downstream targets. The investigation of host AMPK protein modulation during Leishmania infection should primarily be assessed during in vitro infections using as a host murine bone marrow-derived macrophages (BMMos). The infection outcome is assessed measuring the percentage of infected cells in the context of BMMos. To evaluate AMPK activity during infection, the expression of AMPK phosphorylated at Thr172 as well as the transcription and translational levels of its downstream targets are evaluated by quantitative PCR and immunoblotting. The modulation of AMPK activity in vivo is determined specifically in sorted splenic macrophages harboring Leishmania parasites recovered from infected mice using fluorescent-labeled parasites in the infectious inocolum. The modulation of AMPK activity was assessed by AMPK activators and inhibitors and also using AMPK, SIRT1, or LKB1 KO mice models. The infection outcome in BMMos and in vivo was further determined using these two different approaches. To finally understand the metabolic impact of AMPK during infection, in vitro metabolic assays in infected BMMos were measured in the bioenergetic profile using an extracellular flux analyzer.Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contract IF/00021/2014 to R.S.), by FEDER funds through the Operational Competitiveness Programme (COMPETE), and by national funds through FCT (Fundação para a Ciência e a Tecnologia) under the project FCOMP-01-0124-FEDER-011054 (PTDC/SAU-FCF/100749/2008) and PTDC/BIA-MIC/118644/2010. The research leading to these results has also received funding from the European community’s Seventh Framework Programme under grant agreement No.602773 to JE and ACS (Project KINDRED). DM was supported by SFRH/BD/91543/2012. JE thanks the Canada Research Chair program for their support

The influence of surface modified poly(L-lactic acid) films on the differentiation of human monocytes into macrophages

Macrophages play a crucial role in the biological performance of biomaterials, as key factors in defining the optimal inflammation-healing balance towards tissue regeneration and implant integration. Here, we investigate how different surface modifications performed on poly(L-lactic acid) (PLLA) films would influence the differentiation of human monocytes into macrophages. We tested PLLA films without modification, surface-modified by plasma treatment (pPLLA) or by combining plasma treatment with different coating materials, namely poly(L-lysine) and a series of proteins from the extracellular matrix: collagen I, fibronectin, vitronectin, laminin and albumin. While all the tested films are non-cytotoxic, differences in cell adhesion and morphology are observed. Monocyte-derived macrophages (MDM) present a more rounded shape in non-modified films, while a more elongated phenotype is observed containing filopodia-like and podosome-like structures in all modified films. No major differences are found for the expression of HLA-DR+/CD80(+) and CD206(+)/CD163(+) surface markers, as well as for the ability of MDM to phagocytize. Interestingly, MDM differentiated on pPLLA present the highest expression of MMP9. Upon differentiation, MDM in all surface modified films present lower amounts of IL-6 and IL-10 compared to non-modified films. After stimulating MDM with the potent pro-inflammatory agent LPS, pPLLA and poly(L-lysine) and fibronectin-modified films reveal a significant reduction in IL-6 secretion, while the opposite effect is observed with IL-10. Of note, in comparison to non-modified films, all surface modified films induce a significant reduction of the IL-6/IL-10 ratio, a valuable prognosticator of the pro-versus anti-inflammatory balance. These findings provide important insights into MDM-biomaterial interactions, while strengthening the need for designing immune-informed biomaterials.project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER). C. R. Correia and J. F. Mano acknowledge the funding from the European Research Council for project ATLAS with the grant agreement number ERC-2014-ADG-669858. J. Gaifem, M.B. Oliveira and R. Silvestre acknowledge the Portuguese Foundation for Science and Technology (FCT) for the doctoral (PD/BD/106053/2015), post-doctoral (SFRH/BPD/111354/2015) and FCT Investigator (IF/00021/2014) grants, respectively. The authors also acknowledge Hospital de Braga for providing the buffy coatsThis work was developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER). C. R. Correia and J. F. Mano acknowledge the funding from the European Research Council for project ATLAS with the grant agreement number ERC-2014-ADG-669858. J. Gaifem, M.B. Oliveira and R. Silvestre acknowledge the Portuguese Foundation for Science and Technology (FCT) for the doctoral (PD/BD/106053/2015), post-doctoral (SFRH/BPD/111354/2015) and FCT Investigator (IF/00021/2014) grants, respectively. The authors also acknowledge Hospital de Braga for providing the buffy coats.info:eu-repo/semantics/publishedVersio

Analysis of salivary levels of IL-1β, IL17A, OPG and RANK-L in periodontitis using the 2017 Classification of Periodontal Diseases - an exploratory observational study

Periodontitis is a chronic disease with a high overall prevalence. It involves a complex interplay between the immune-inflammatory pathways and biofilm changes, leading to periodontal attachment loss. The aims of this study were (i) to assess whether the salivary IL-1β, IL-17A, RANK-L and OPG levels have the potential to discriminate between the mild and severe periodontitis conditions; and (ii) to enable diagnostic/prognostic actions to differentiate between distinct levels of the disease. The analysis of the clinical parameters and the evaluation of the salivary immunomediators levels by means of a multiplex flow assay revealed a statistically significantly higher level of IL-1β in the periodontitis III/IV patients, as well as a higher level of RANK-L in the periodontitis III/IV and I/II patients, when compared to the healthy controls. Furthermore, the grade C periodontitis patients presented a significantly higher level of RANK-L compared to the grade B and grade A patients. In the grade C patients, IL-1β had a positive correlation with the PPD and CAL indices and RANK_L had a positive correlation with CAL. The evidence emerging from this study associates the salivary IL-1β and RANK-L levels with an advanced stage of periodontitis, stage III/IV, and with grade C, suggesting the possible cooperative action of both in the inflammatory and bone loss events. In addition to IL-1β, RANK-L could be considered a combined diagnostic biomarker for periodontitis.This research was funded by the University Institute of Health Sciences (IUCS-CESPU). The participation of Marta Relvas was funded by the project grants AMDNCPD_PI2RL_IINFACTS_2021 and ADMT1PD_GI2-CESPU_2022, while the participation of Luis Monteiro was funded by the project grant mTORORAL_GI2-CESPU_2022 from CESPU University, by the Foundation for Science and Technology (FCT)–project UIDB/50026/2020 and UIDP/50026/2020, by the NORTE-01-0145- FEDER-000013 and NORTE-01-0145-FEDER-000023 projects, and by the Norte Portugal Regional Operational Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF), the FCT contracts 2021.07836.BD to AMF and CEECIND/00185/2020 to RS

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies.This work was supported by CHIR (Canada) and ANRS grants (France). JE thanks the Canada Research Chair program for financial assistance. VR was supported by a doctoral fellowship from FCT (Fundacao para a Ciencia e Tecnologia); code SFRH/BD/64064/2009. We would like to thank the Nonhuman Primate Reagent Resource for kindly providing CXCR5 antibodies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The impact of IL-10 dynamic modulation on host immune response against visceral leishmaniasis

Leishmaniasis is a vector-borne disease caused by protozoan parasites from the genus Leishmania. The most severe form of disease is visceral leishmaniasis (VL), which is fatal if left untreated. It has been demonstrated that interleukin (IL)-10, is associated with disease progression and susceptibility. In this work, we took advantage of a transgenic mouse model that expresses high levels of IL-10 upon zinc sulfate administration (pMT-10). We addressed the role of IL-10 during the initial stages of L. donovani infection by analyzing the parasite burden in the spleen and liver of the infected pMT-10 and WT mice as well as the histopathological alterations upon IL-10 induction. Furthermore, the profile of cytokines expressed by T cells was assessed. Our results demonstrate that an increase in IL-10 production has an impact early but not later after infection. This specific temporal role for IL-10-mediated susceptibility to VL is of interest.Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contracts SFRH/BD/120127/2016 to IM, PD/BDE/127830/2016 to CF, SFRH/BD/120371/2016 to AMB, IF/01147/2013 to RDO, IF/01390/2014 to ET, IF/00735/2014 to AC, SFRH/BPD/96176/2013 to CC and IF/00021/2014 to RS), and Infect-Era (project INLEISH). JE also thanks the Canada Research Chair program for financial assistanceinfo:eu-repo/semantics/publishedVersio

The Warburg effect in mycobacterial granulomas is dependent on the recruitment and activation of macrophages by interferon-γ

Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon-y (IFN-y). Mycobacterium avium-infected mice lacking IFN-y signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine-18-fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN-y signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN-y reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN-y displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN-y is responsible for the Warburg effect observed in organs infected with M. avium.Funded by project ‘NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions’ co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER) and by Fundação para a Ciência e Tecnologia