Biomedical applications of human cathelicidin

[Excerpt] Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, represents the second world’s deadliest infectious disease, affecting around 9 million people worldwide in 2013. Of those, about 1.1 million died from the disease. The potential of cathelicin, a human AMP, in the treatment of mycobacteriosis and wound regeneration was assessed in pre-clinical trials. (...

Endogenous cathelicidin production limits inflammation and protective immunity to Mycobacterium avium in mice

The production of antimicrobial peptides, such as the cathelicidins, plays a prominent role in the innate immune response against microbial pathogens. Cathelicidins are widely distributed amongst living organisms, and the antimicrobial peptides generated by proteolysis of the precursor forms are typically cationic and [alpha]-helical, a structure that facilitates their interaction and insertion into anionic bacterial cell walls and membranes, causing damage and promoting microbial death. Here, we found that mouse cathelicidin (Camp) expression was induced in bone marrow-derived macrophages by infection with Mycobacterium avium in a TLR2- and TNF-dependent manner. However, the endogenous production of the cathelin-related antimicrobial peptide (CRAMP) was not required for the bacteriostasis of M. avium either in primary cultures of macrophages or in vivo, as shown by the use of CRAMP-null mice. In contrast, the lack of Camp led to a transient improvement of M. avium growth control in the spleens of infected mice while at the same time causing an exacerbation of the inflammatory response to infection. Our data highlight the anti-inflammatory effects of CRAMP and suggests that virulent mycobacteria may possess strategies to escape its antimicrobial activity.Funded through project PTDC/BIA-BCM/112138/2009FCOMP-01-0124-FEDER014185

Delivery of antimicrobial peptides for the treatment of mycobacteriosis

Mycobacterium tuberculosis, which resides inside macrophages, has always been recognized as one of the most “successful” pathogens. Standard treatments have already been used for decades and, therefore, resistances to the first-line medicines are increasing. Additionally, poor patient compliance with stringent therapies is often pointed out as a major reason leading to treatment failure. Antimicrobial peptides (AMPs), a promising new class of broad spectrum antibiotics, are less prone to result in pathogen resistances due to their target (cellular membranes) and rapid action. In our laboratory we search for AMPs with potent activity against mycobacteria and try to develop efficient delivery systems based on self-assembled colloidal nanocarriers. Additionally, this systems are expected to reduce peptide toxicity and enhance selective uptake on infected cells. Finally, the use of encapsulated drugs in mycobacterial therapy may help reducing drug administration schedules which would ultimately improve patient compliance

Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)-and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.

Various approaches to the synthesis of all four stereoisomers of 2-(1H- imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct transcyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine

Delivery of LLKKK18 loaded into self-assembling hyaluronic acid nanogel for tuberculosis treatment

uberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, recently joined HIV/AIDS on the top rank of deadliest infectious diseases. Low patient compliance due to the expensive, long-lasting and multi-drug standard therapies often results in treatment failure and emergence of multi-drug resistant strains. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment. Here we describe the ability of the exogenous AMP LLKKK18 to efficiently kill mycobacteria. The peptide's potential was boosted by loading into self-assembling Hyaluronic Acid (HA) nanogels. These provide increased stability, reduced cytotoxicity and degradability, while potentiating peptide targeting to main sites of infection. The nanogels were effectively internalized by macrophages and the peptide presence and co-localization with mycobacteria within host cells was confirmed. This resulted in a significant reduction of the mycobacterial load in macrophages infected in vitro with the opportunistic M. avium or the pathogenic M. tuberculosis, an effect accompanied by lowered pro-inflammatory cytokine levels (IL-6 and TNF-). Remarkably, intra-tracheal administration of peptide-loaded nanogels significantly reduced infection levels in mice infected with M. avium or M. tuberculosis, after just 5 or 10 every other day administrations. Considering the reported low probability of resistance acquisition, these findings suggest a great potential of LLKKK18-loaded nanogels for TB therapeutics.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER- 006684). The authors also acknowledge the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors thank Dr. Hugo Osório (Proteomics Lab at I3S – Institute for Health Research and Innovation, Porto, Portugal) for the MALDI-ToF analysis. JPS acknowledges FCT for the financial support provided by grant SFRH/BPD/64958/2010

Delivery of nanogel formulations with antimicrobial peptides for the treatment of mycobacteriosis

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Mycobacterium tuberculosis is the human pathogen that causes Tuberculosis (TB). In 2015, 10.4 million TB cases and 1.8 million deaths were reported, placing this disease alongside HIV/AIDS as the deadliest infectious diseases. Current treatments rely in the administration of a cocktail of four first-line antibiotics during 6 months and, in the worst case scenario, a long-lasting treatment (24 months) with second-line drugs. The overuse or misuse of antimicrobial agents decreases the success of treatments and increases emergence of Multi-drug resistant (MDR) strains. Therefore, the development of new strategies for TB therapy is urgently needed. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment since they present high spectrum of antimicrobial activity, high efficacy at low concentrations and low propensity for bacterial resistance. Nevertheless, the low capacity of AMPs to reach the infected site and the use of high concentrations to overcome this problem limits its clinical application - this can be circumvented using a drug delivery system [1]. [...]info:eu-repo/semantics/publishedVersio

T Cells home to the thymus and control infection

The thymus is a target of multiple pathogens. How the immune system responds to thymic infection is largely unknown. Despite being considered an immune-privileged organ, we detect a mycobacteria-specific T cell response in the thymus following dissemination of Mycobacterium avium or Mycobacterium tuberculosis. This response includes proinflammatory cytokine production by mycobacteria-specific CD4(+) and CD8(+) T cells, which stimulates infected cells and controls bacterial growth in the thymus. Importantly, the responding T cells are mature peripheral T cells that recirculate back to the thymus. The recruitment of these cells is associated with an increased expression of Th1 chemokines and an enrichment of CXCR3(+) mycobacteria-specific T cells in the thymus. Finally, we demonstrate it is the mature T cells that home to the thymus that most efficiently control mycobacterial infection. Although the presence of mature T cells in the thymus has been recognized for some time, to our knowledge, these data are the first to show that T cell recirculation from the periphery to the thymus is a mechanism that allows the immune system to respond to thymic infection. Maintaining a functional thymic environment is essential to maintain T cell differentiation and prevent the emergence of central tolerance to the invading pathogens.This work was supported by Portuguese Foundation for Science and Technology Grant PTDC/SAU-MII/101663/2008 and individual fellowships to C.N., C.N.-A., B.C.-R., S.R., and P.B.-S. S.M.B. was supported by National Institutes of Health Grant R01 AI067731. The Small Animal Biocontainment Suite was supported in part by Center for AIDS Research Grant P30 AI 060354

Mycobacteria-induced anaemia revisited : a molecular approach reveals the involvement of NRAMP1 and lipocalin-2, but not of hepcidin

Anaemia is a frequent complication of chronic infectious diseases but the exact mechanisms by which it develops remain to be clarified. In the present work, we used a mouse model of mycobacterial infection to study molecular alterations of iron metabolism induced by infection. We show that four weeks after infection with Mycobacterium avium BALB/c mice exhibited a moderate anaemia, which was not accompanied by an increase on hepatic hepcidin mRNA expression. Instead, infected mice presented increased mRNA expression of ferroportin (Slc40a1), ceruloplasmin (Cp), hemopexin (Hpx), heme-oxygenase-1 (Hmox1) and lipocalin-2 (Lcn2). Both the anaemia and the mRNA expression changes of iron-related genes were largely absent in C.D2 mice which bear a functional allele of the Nramp1 gene. Data presented in this work suggest that anaemia due to a chronic mycobacterial infection may develop in the absence of elevated hepcidin expression, is influenced by Nramp1 and may involve lipocalin-2.This work was supported by the EEC Framework 6 (LSHM-CT-2006037296 EuroIron1) and FCT-approved grant POCTI/MGI/40132/2001, funded by FEDER. Sandro Gomes was supported by FCT PhD grant SFRH/BD/29257/2006

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

Studies in the mouse model identify strain variability as a major determinant of disease outcome in Leishmania infantum infection

Visceral leishmaniasis is a severe and potentially fatal disease caused by protozoa of the genus Leishmania, transmitted by phlebotomine sandflies. In Europe and the Mediterranean region, L. infantum is the commonest agent of visceral leishmaniasis, causing a wide spectrum of clinical manifestations, including asymptomatic carriage, cutaneous lesions and severe visceral disease. Visceral leishmaniasis is more frequent in immunocompromised individuals and data obtained in experimental models of infection have highlighted the importance of the host immune response, namely the efficient activation of host's macrophages, in determining infection outcome. Conversely, few studies have addressed a possible contribution of parasite variability to this outcome.No funders or funding refered in the paper