Hostname: page-component-78c5997874-dh8gc Total loading time: 0 Render date: 2024-11-19T09:19:42.594Z Has data issue: false hasContentIssue false
  • English
  • Français

Ageing impairs protective immunity and promotes susceptibility to murine visceral leishmaniasis

Published online by Cambridge University Press:  07 June 2022

Caio Loureiro Salgado ,
Andrés Felipe Mendéz Corea ,
Luciana Polaco Covre ,
Herbet Leonel De Matos Guedes Open the ORCID record for Herbet Leonel De Matos Guedes [Opens in a new window] ,
Aloisio Falqueto  and
Daniel Cláudio Oliviera Gomes Open the ORCID record for Daniel Cláudio Oliviera Gomes [Opens in a new window]
Caio Loureiro Salgado
Affiliation:
Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitoria, Brazil
Andrés Felipe Mendéz Corea
Affiliation:
Núcleo de Biotecnologia, Universidade Federal do Espírito Santo, Vitoria, Brazil
Luciana Polaco Covre
Affiliation:
Division of Medicine, University College London, London, UK
Herbet Leonel De Matos Guedes
Affiliation:
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Aloisio Falqueto
Affiliation:
Departamento de Medicina Social, Universidade Federal do Espírito Santo, Vitoria, Brazil
Daniel Cláudio Oliviera Gomes*
Affiliation:
Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitoria, Brazil Núcleo de Biotecnologia, Universidade Federal do Espírito Santo, Vitoria, Brazil
*
Author for correspondence: Daniel Cláudio Oliviera Gomes, E-mail: dgomes@ndi.ufes.br
Get access Rights & Permissions [Opens in a new window]

Abstract

It is well accepted that the impact of diseases is generally more detrimental in elderly individuals than in younger ones. Changes in the immune system due to ageing can directly affect the ability to respond effectively to infections and may contribute to the higher morbidities and mortalities in the elderly population. Leishmaniasis is a complex of clinically unique diseases caused by obligate intracellular protozoa belonging to genus Leishmania, wherein visceral leishmaniasis (VL) is the most severe form and is fatal if left untreated. In this study, aged mice (72 weeks old) presented increased susceptibility to L. infantum infection compared to younger mice (4–6-week-old), with notable parasitism in both the spleen and liver, as well as exhibiting hepatosplenomegaly. A pronounced inflammatory profile was observed in the aged-infected mice, with excessive production of TNF-α and nitrite, along with diminished IFN-γ production and reduced proliferative capacity of T cells (assessed by expression of the Ki67 marker). Additionally, both CD4+ and CD8+ T cells from the aged-infected mice presented increased expression of the inhibitory receptors PD-1 and KLRG1 that strongly correlated with the parasitism found in the liver and spleen of this group. Overall, the data reported in this study suggests for the first time that ageing may negatively impact the VL outcome and provides a perspective for new therapeutic strategies involving manipulation of immunosenescence features against Leishmania infection.

Keywords

InflammationLeishmania infantumsenescencevisceral leishmaniasis
Type
Research Article
Information
Parasitology , Volume 149 , Issue 9 , August 2022 , pp. 1249 - 1256
Check for updates
Copyright
Copyright © The Author(s), 2022. Published by Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Aiello, A, Farzaneh, F, Candore, G, Caruso, C, Davinelli, S, Gambino, CM, Ligotti, ME, Zareian, N and Accardi, G (2019) Immunosenescence and its hallmarks: how to oppose aging strategically? A review of potential options for therapeutic intervention. Frontiers in Immunology 10, 2247.CrossRefGoogle ScholarPubMed
Akbar, AN, Henson, SM and Lanna, A (2016) Senescence of T lymphocytes: implications for enhancing human immunity. Trends in Immunology 37, 1–11.CrossRefGoogle ScholarPubMed
Alvar, J, Vélez, ID, Bern, C, Herrero, M, Desjeux, P, Cano, J, Jannin, J and de Boer, M (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7, e35671.CrossRefGoogle ScholarPubMed
Araujo-Melo, MH, Meneses, AM, Schubach, AO, Moreira, JS, Conceição-Silva, F, Salgueiro, MM, Pimentel, MIF, Araújo-Silva, M, Oliveira, RVC, Carmo, CN and Valete-Rosalino, CM (2010) Risk factors associated with dizziness during treatment of mucosal leishmaniasis with meglumine antimoniate: 16-year retrospective study of cases from Rio de Janeiro, Brazil. The Journal of Laryngology and Otology 124, 1056–1060.CrossRefGoogle ScholarPubMed
Barbé-Tuana, F, Funchal, G, Schmitz, CRR, Maurmann, RM and Bauer, ME (2020) The interplay between immunosenescence and age-related diseases. Seminars in Immunopathology 42, 545–557.CrossRefGoogle ScholarPubMed
Bender, BS, Johnson, MP and Small, PA (1991) Influenza in senescent mice: impaired cytotoxic T-lymphocyte activity is correlated with prolonged infection. Immunology 72, 514–519.Google ScholarPubMed
Bogdan, C (2020) Macrophages as host, effector and immunoregulatory cells in leishmaniasis: impact of tissue micro-environment and metabolism. Cytokine: X 2, 100041.Google ScholarPubMed
Burza, S, Croft, SL and Boelaert, M (2018) Leishmaniasis. The Lancet 392, 951–970.CrossRefGoogle ScholarPubMed
Carvalho, LP, Passos, S, Bacellar, O, Lessa, M, Almeida, RP, Magalhães, A, Dutra, WO, Gollob, KJ, Machado, P and Ribeiro De Jesus, A (2007) Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis. Parasite Immunology 29, 251–258.CrossRefGoogle ScholarPubMed
Cerqueira, É, Marinho, DA, Neiva, HP and Lourenço, O (2020) Inflammatory effects of high and moderate intensity exercise – a systematic review. Frontiers in Physiology 10, 1550.CrossRefGoogle ScholarPubMed
Chen, J, Kelley, WJ and Goldstein, DR (2020) Role of aging and the immune response to respiratory viral infections: potential implications for COVID-19. Journal of Immunology (Baltimore, MD: 1950) 205, 313–320.CrossRefGoogle ScholarPubMed
Childs, BG, Durik, M, Baker, DJ and van Deursen, JM (2015) Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nature Medicine 21, 1424–1435.CrossRefGoogle ScholarPubMed
Cincurá, C, de Lima, CMF, Machado, PRL, Oliveira-Filho, J, Glesby, MJ, Lessa, MM and Carvalho, EM (2017) Mucosal leishmaniasis: a retrospective study of 327 cases from an endemic area of Leishmania (Viannia) braziliensis. The American Journal of Tropical Medicine and Hygiene 97, 761–766.CrossRefGoogle ScholarPubMed
Coppé, J-P, Desprez, P-Y, Krtolica, A and Campisi, J (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annual Review of Pathology 5, 99–118.CrossRefGoogle ScholarPubMed
Covre, LP, Martins, RF, Devine, OP, Chambers, ES, Vukmanovic-Stejic, M, Silva, JA, Dietze, R, Rodrigues, RR, de Matos Guedes, HL, Falqueto, A, Akbar, AN and Gomes, DCO (2019) Circulating senescent T cells are linked to systemic inflammation and lesion size during human cutaneous leishmaniasis. Frontiers in Immunology 9, 3001.CrossRefGoogle ScholarPubMed
da Fonseca-Martins, AM, Ramos, TD, Pratti, JES, Firmino-Cruz, L, Gomes, DCO, Soong, L, Saraiva, EM and de Matos Guedes, HL (2019) Immunotherapy using anti-PD-1 and anti-PD-L1 in Leishmania amazonensis-infected BALB/c mice reduce parasite load. Scientific Reports 9, 20275.CrossRefGoogle ScholarPubMed
de Freitas e Silva, R and von Stebut, E (2021) Unraveling the role of immune checkpoints in leishmaniasis. Frontiers in Immunology 12, 1664–3224.CrossRefGoogle ScholarPubMed
de Lima, VMF, Peiro, JR and de Oliveira Vasconcelos, R (2007) IL-6 and TNF-alpha production during active canine visceral leishmaniasis. Veterinary Immunology and Immunopathology 115, 189–193.CrossRefGoogle ScholarPubMed
de Moura, R, Covre, LP, Fantecelle, CH, Gajardo, VAT, Cunha, CB, Stringari, LL, Belew, AT, Daniel, CB, Von Zeidler, SV, Tadokoro, CE, de Matos Guedes, HL, Zanotti, RL, Mosser, D, Falqueto, A, Akbar, AN and Gomes, DCO (2021) PD-1 blockade modulates functional activities of exhausted-like T cell in patients with cutaneous leishmaniasis. Frontiers in Immunology 12, 1664–3224.Google Scholar
Diniz, DS, Costa, ASV and Escalda, PMF (2012) The effect of age on the frequency of adverse reactions caused by antimony in the treatment of American tegumentary leishmaniasis in Governador Valadares, State of Minas Gerais, Brazil. Revista da Sociedade Brasileira de Medicina Tropical 45, 597–600.CrossRefGoogle ScholarPubMed
Ehrchen, J, Sindrilaru, A, Grabbe, S, Schönlau, F, Schlesiger, C, Sorg, C, Scharffetter-Kochanek, K and Sunderkötter, C (2004) Senescent BALB/c mice are able to develop resistance to Leishmania major infection. Infection and Immunity 72, 5106–5114.CrossRefGoogle ScholarPubMed
Fang, M, Roscoe, F and Sigal, LJ (2010) Age-dependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking. The Journal of Experimental Medicine 207, 2369–2381.CrossRefGoogle ScholarPubMed
Faria, DR, Gollob, KJ, Barbosa, J, Schriefer, A, Machado, PRL, Lessa, H, Carvalho, LP, Romano-Silva, MA, De Jesus, AR, Carvalho, EM and Dutra, WO (2005) Decreased in situ expression of interleukin-10 receptor is correlated with the exacerbated inflammatory and cytotoxic responses observed in mucosal leishmaniasis. Infection and Immunity 73, 7853–7859.CrossRefGoogle ScholarPubMed
Freund, A, Orjalo, AV, Desprez, P-Y and Campisi, J (2010) Inflammatory networks during cellular senescence: causes and consequences. Trends in Molecular Medicine 16, 238–246.CrossRefGoogle ScholarPubMed
Gardner, ID (1980) The effect of aging on susceptibility to infection. Reviews of Infectious Diseases 2, 801–810.CrossRefGoogle ScholarPubMed
Gasser, S, Orsulic, S, Brown, EJ and Raulet, DH (2005) The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 436, 1186–1190.CrossRefGoogle ScholarPubMed
Green, LC, Wagner, DA, Glogowski, J, Skipper, PL, Wishnok, JS and Tannenbaum, SR (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical Biochemistry 126, 131–138.CrossRefGoogle Scholar
Groh, V, Rhinehart, R, Randolph-Habecker, J, Topp, MS, Riddell, SR and Spies, T (2001) Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nature Immunology 2, 255–260.CrossRefGoogle ScholarPubMed
Horta, MF, Mendes, BP, Roma, EH, Noronha, FSM, Macêdo, JP, Oliveira, LS, Duarte, MM and Vieira, LQ (2012) Reactive oxygen species and nitric oxide in cutaneous leishmaniasis. Journal of Parasitology Research 2012, 203818.CrossRefGoogle ScholarPubMed
Jirmanus, L, Glesby, MJ, Guimarães, LH, Lago, E, Rosa, ME, Machado, PR and Carvalho, EM (2012) Epidemiological and clinical changes in American tegumentary leishmaniasis in an area of Leishmania (Viannia) braziliensis transmission over a 20-year period. The American Journal of Tropical Medicine and Hygiene 86, 426–433.CrossRefGoogle Scholar
Krone, CL, Trzciński, K, Zborowski, T, Sanders, EAM and Bogaert, D (2013) Impaired innate mucosal immunity in aged mice permits prolonged Streptococcus pneumoniae colonization. Infection and Immunity 81, 4615–4625.CrossRefGoogle ScholarPubMed
Lanna, A, Gomes, DCO, Muller-Durovic, B, McDonnell, T, Escors, D, Gilroy, DW, Lee, JH, Karin, M and Akbar, AN (2017) A sestrin-dependent Erk–Jnk–p38 MAPK activation complex inhibits immunity during aging. Nature Immunology 18, 354–363.CrossRefGoogle ScholarPubMed
Larbi, A, Pawelec, G, Wong, SC, Goldeck, D, Tai, JJ-Y and Fulop, T (2011) Impact of age on T cell signaling: a general defect or specific alterations? Ageing Research Reviews 10, 370–378.CrossRefGoogle ScholarPubMed
Leal, JM, Mosquini, M, Covre, LP, Stagmiller, NP, Rodrigues, RR, Christensen, D, Guedes, HLDM, Rossi-Bergmann, B and Gomes, DCDO (2015) Intranasal vaccination with killed Leishmania amazonensis promastigotes antigen (LaAg) associated with CAF01 adjuvant induces partial protection in BALB/c mice challenged with Leishmania (infantum) chagasi. Parasitology 142, 1640–1646.CrossRefGoogle ScholarPubMed
Leng, J, Stout-Delgado, HW, Kavita, U, Jacobs, A, Tang, J, Du, W, Tussey, L and Goldstein, DR (2011) Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza viral infection. Vaccine 29, 8147–8155.CrossRefGoogle ScholarPubMed
Liang, SC, Greenwald, RJ, Latchman, YE, Rosas, L, Satoskar, A, Freeman, GJ and Sharpe, AH (2006) PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis. European Journal of Immunology 36, 58–64.CrossRefGoogle ScholarPubMed
Liew, FY, Li, Y and Millott, S (1990) Tumour necrosis factor (TNF-alpha) in leishmaniasis. II. TNF-alpha-induced macrophage leishmanicidal activity is mediated by nitric oxide from L-arginine. Immunology 71, 556–559.Google ScholarPubMed
Marston, BJ, Plouffe, JF, File, TM Jr., Hackman, BA, Salstrom, S-J, Lipman, HB, Kolczak, MS and Breiman, RF (1997) Incidence of community-acquired pneumonia requiring hospitalization: results of a population-based active surveillance study in Ohio. Archives of Internal Medicine 157, 1709–1718.CrossRefGoogle ScholarPubMed
Mauch, P, Botnick, LE, Hannon, EC, Obbagy, J and Hellman, S (1982) Decline in bone marrow proliferative capacity as a function of age. Blood 60, 245–252.CrossRefGoogle ScholarPubMed
Müller-Durovic, B, Lanna, A, Polaco, L, Mills, RS, Henson, SM and Akbar, AN (2016) Killer cell lectin-like receptor G1 inhibits NK cell function through activation of adenosine 5′-monophosphate – activated protein kinase. The Journal of Immunology (Baltimore, MD: 1950) 197, 2891–2899.CrossRefGoogle ScholarPubMed
Murasko, DM and Jiang, J (2005) Response of aged mice to primary virus infections. Immunological Reviews 205, 285–296.CrossRefGoogle ScholarPubMed
Norman, DC (2016) Clinical features of infection in older adults. Clinics in Geriatric Medicine 32, 433–441.CrossRefGoogle ScholarPubMed
Oliveira, F, Bafica, A, Rosato, AB, Favali, CBF, Costa, JM, Cafe, V, Barral-Netto, M and Barral, A (2011) Lesion size correlates with Leishmania antigen-stimulated TNF-levels in human cutaneous leishmaniasis. The American Journal of Tropical Medicine and Hygiene 85, 70–73.CrossRefGoogle ScholarPubMed
Oliveira, AGL, Brito, PD, Schubach, AO, Oliveira, RVC, Saheki, MN, Lyra, MR, Salgueiro, MM, Terceiro, BF, Pimentel, MIF, Vasconcellos, EC and Valete-Rosalino, CM (2013) Influence of the nutritional status in the clinical and therapeutical evolution in adults and elderly with American Tegumentary Leishmaniasis. Acta Tropica 128, 36–40.CrossRefGoogle ScholarPubMed
Oliveira Silva, KL, Marin Chiku, V, Luvizotto Venturin, G, Correa Leal, AA, de Almeida, BF, De Rezende Eugenio, F, Dos Santos, PSP, Fabrino Machado, G and De Lima, VMF (2019) PD-1 and PD-L1 regulate cellular immunity in canine visceral leishmaniasis. Comparative Immunology, Microbiology and Infectious Diseases 62, 76–87.CrossRefGoogle ScholarPubMed
Pavlicek, D, Krebs, J, Capossela, S, Bertolo, A, Engelhardt, B, Pannek, J and Stoyanov, J (2017) Immunosenescence in persons with spinal cord injury in relation to urinary tract infections – a cross-sectional study. Immunity & Ageing: I & A 14, 22.CrossRefGoogle ScholarPubMed
Plunkett, FJ, Franzese, O, Finney, HM, Fletcher, JM, Belaramani, LL, Salmon, M, Dokal, I, Webster, D, Lawson, ADG and Akbar, AN (2007) The loss of telomerase activity in highly differentiated CD8+CD28-CD27- T cells is associated with decreased Akt (Ser473) phosphorylation. The Journal of Immunology (Baltimore, MD: 1950) 178, 7710–7719.CrossRefGoogle ScholarPubMed
Ponnappan, S and Ponnappan, U (2011) Aging and immune function: molecular mechanisms to interventions. Antioxidants & Redox Signaling 14, 1551–1585.CrossRefGoogle Scholar
Prata, LGPL, Ovsyannikova, IG, Tchkonia, T and Kirkland, JL (2018) Senescent cell clearance by the immune system: emerging therapeutic opportunities. Seminars in Immunology 40, 101275.CrossRefGoogle ScholarPubMed
Rebel, VI, Miller, CL, Eaves, CJ and Lansdorp, PM (1996) The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 87, 3500–3507.CrossRefGoogle ScholarPubMed
Reiner, SL and Locksley, RM (1995) The regulation of immunity to Leishmania major. Annual Review of Immunology 13, 151–177.CrossRefGoogle ScholarPubMed
Rodrigues, V, Laforge, M, Campillo-Gimenez, L, Soundaramourty, C, Correia-de-Oliveira, A, Dinis-Oliveira, RJ, Ouaissi, A, Cordeiro-da-Silva, A, Silvestre, R and Estaquier, J (2014) Abortive T follicular helper development is associated with a defective humoral response in Leishmania infantum-infected macaques. PLOS Pathogens 10, e1004096.CrossRefGoogle ScholarPubMed
Rodrigues, V, Cordeiro-Da-Silva, A, Laforge, M, Silvestre, R and Estaquier, J (2016) Regulation of immunity during visceral Leishmania infection. Parasites and Vectors 9, 118.CrossRefGoogle ScholarPubMed
Rufer, N, Brümmendorf, TH, Kolvraa, S, Bischoff, C, Christensen, K, Wadsworth, L, Schulzer, M and Lansdorp, PM (1999) Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. Journal of Experimental Medicine 190, 157–168.CrossRefGoogle Scholar
Scott, P (1991) IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. The Journal of Immunology (Baltimore, MD: 1950) 147, 3149–3155.Google Scholar
Scott, P, Pearce, E, Cheever, AW, Coffman, RL and Sher, A (1989) Role of cytokines and CD4+ T-cell subsets in the regulation of parasite immunity and disease. Immunological Reviews 112, 161–182.CrossRefGoogle ScholarPubMed
Solana, R, Tarazona, R, Gayoso, I, Lesur, O, Dupuis, G and Fulop, T (2012) Innate immunosenescence: effect of aging on cells and receptors of the innate immune system in humans. Seminars in Immunology 24, 331–341.CrossRefGoogle ScholarPubMed
Sorci, G, Léchenault-Bergerot, C and Faivre, B (2021) Age reduces resistance and tolerance in malaria-infected mice. Infection, Genetics and Evolution 88, 104698.CrossRefGoogle ScholarPubMed
Speziali, E, Aranha, CHM, Teixeira-Carvalho, A, Santiago, AF, Oliveira, RP, Rezende, MC, Carneiro, CM, Negrão-Corrêa, D, Coelho, PMZ and Faria, AMC (2010) Ageing down-modulates liver inflammatory immune responses to schistosome infection in mice. Scandinavian Journal of Immunology 71, 240–248.CrossRefGoogle ScholarPubMed
Squires, KE, Schreiber, RD, McElrath, MJ, Rubin, BY, Anderson, SL and Murray, HW (1989) Experimental visceral leishmaniasis: role of endogenous IFN-gamma in host defense and tissue granulomatous response. The Journal of Immunology (Baltimore, MD: 1950) 143, 4244–4249.Google ScholarPubMed
Toapanta, FR and Ross, TM (2009) Impaired immune responses in the lungs of aged mice following influenza infection. Respiratory Research 10, 112.CrossRefGoogle ScholarPubMed
Torres-Guerrero, E, Quintanilla-Cedillo, MR, Ruiz-Esmenjaud, J and Arenas, R (2017) Leishmaniasis: a review. F1000Research 6, 750.CrossRefGoogle ScholarPubMed
Tripathi, P, Singh, V and Naik, S (2007) Immune response to leishmania: paradox rather than paradigm. FEMS Immunology and Medical Microbiology 51, 229–242.CrossRefGoogle ScholarPubMed
Tsuji, S, Minami, S, Hashimoto, R, Konishi, Y, Suzuki, T, Kondo, T, Sasai, M, Torii, S, Ono, C, Shichinohe, S, Sato, S, Wakita, M, Okumura, S, Nakano, S, Matsudaira, T, Matsumoto, T, Kawamoto, S, Yamamoto, M, Watanabe, T, Matsuura, Y, Takayama, K, Kobayashi, T, Okamoto, T and Hara, E (2022) SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response. Nature Aging 2, 115–124.CrossRefGoogle Scholar
Wang, JM, Cheng, YQ, Shi, L, Ying, RS, Wu, XY, Li, GY, Moorman, JP and Yao, ZQ (2013) KLRG1 negatively regulates natural killer cell functions through the Akt pathway in individuals with chronic hepatitis C virus infection. Journal of Virology 87, 11626–11636.CrossRefGoogle ScholarPubMed
Weinberger, B, Herndler-Brandstetter, D, Schwanninger, A, Weiskopf, D and Grubeck-Loebenstein, B (2008) Biology of immune responses to vaccines in elderly persons. Clinical Infectious Diseases: an Official Publication of the Infectious Diseases Society of America 46, 1078–1084.CrossRefGoogle ScholarPubMed
Williams, AE, José, RJ, Brown, JS and Chambers, RC (2015) Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production. American Journal of Physiology. Lung Cellular and Molecular Physiology 308, L539–L549.CrossRefGoogle ScholarPubMed
Wilson, ME, Jeronimo, SMB and Pearson, RD (2005) Immunopathogenesis of infection with the visceralizing Leishmania species. Microbial Pathogenesis 38, 147–160.CrossRefGoogle ScholarPubMed
Supplementary material: Image

Loureiro Salgado et al. supplementary material

Loureiro Salgado et al. supplementary material 1

Download Loureiro Salgado et al. supplementary material(Image)
Image 91.9 KB
Supplementary material: Image

Loureiro Salgado et al. supplementary material

Loureiro Salgado et al. supplementary material 2

Download Loureiro Salgado et al. supplementary material(Image)
Image 46.6 KB