scholarly journals Immune Response and Protective Efficacy of a Heterologous DNA-Protein Immunization withLeishmaniaSuperoxide Dismutase B1

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Abebe Genetu Bayih ◽  
Nada S. Daifalla ◽  
Lashitew Gedamu
Keyword(s):  
Immune Response ◽  
Mammalian Cells ◽  
Leishmania Donovani ◽  
Protective Efficacy ◽  
Vaccine Antigen ◽  
High Dose ◽  
Spleen Cells ◽  
Th2 Response ◽  
Flow Cytometry Data ◽  
Multiparametric Flow Cytometry

Growing evidence shows that antioxidant proteins ofLeishmaniacould be used as vaccine candidates. In this study, we report the efficacy ofLeishmania donovaniiron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-αor IL-2 by antigen-stimulated T cells. Eight weeks afterL. majorinfection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γproduction by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high doseL. majorinfection.

scholarly journals Differential Immune Response against RecombinantLeishmania donovaniPeroxidoxin 1 and Peroxidoxin 2 Proteins in BALB/c Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Nada S. Daifalla ◽  
Abebe Genetu Bayih ◽  
Lashitew Gedamu
Keyword(s):  
Immune Response ◽  
Leishmania Donovani ◽  
Cpg Odn ◽  
Spleen Cells ◽  
Th2 Response ◽  
Tlr Agonists ◽  
Igg2a Isotype ◽  
High Level ◽  
Th 1 ◽  
Igg1 Isotype

We assessed the immune response against recombinant proteins of two related, albeit functionally different, peroxidoxins fromLeishmania donovani: peroxidoxin 1 (LdPxn1) and peroxidoxin 2 (LdPxn2) in BALB/c mice. We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response. Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype. Antigen-stimulated spleen cells from mice that were immunized with rLdPxn1 produced low level of IL-10 and IL-4 and no IFN-γ, whereas cells from mice immunized with rLdPxn2 secreted high level of IFN-γ, low IL-4, and no IL-10. Coadministration of CpG ODN or GLA-SE with rLdPxn1 skewed the immune response towards a Th 1 type as indicated by robust production of IgG2a isotype. Furthermore, the presence of TLR agonists together with rLdPxn1 antigen enhanced the production of IFN-γand to a lesser extent of IL-10. TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

scholarly journals gp63 in Stable Cationic Liposomes Confers Sustained Vaccine Immunity to Susceptible BALB/c Mice Infected with Leishmania donovani

2008 ◽  
Vol 76 (3) ◽  
pp. 1003-1015 ◽  
Author(s):  
Swati Bhowmick ◽  
Rajesh Ravindran ◽  
Nahid Ali
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Protective Efficacy ◽  
Cationic Liposomes ◽  
Interleukin 4 ◽  
Th2 Response ◽  
Parasitic Burden ◽  
Ifn Γ ◽  
Dose Dependent

ABSTRACT Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 μg of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-γ) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-γ and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8+ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.

scholarly journals The IL-33/ST2 Axis Is Associated with Human Visceral Leishmaniasis and Suppresses Th1 Responses in the Livers of BALB/c Mice Infected with Leishmania donovani

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Octavie Rostan ◽  
Jean-Pierre Gangneux ◽  
Claire Piquet-Pellorce ◽  
Christelle Manuel ◽  
Andrew N. J. McKenzie ◽  
...  
Keyword(s):  
Immune Response ◽  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Parasite Burden ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytokine Signaling ◽  
Th2 Response ◽  
Content Type ◽  
Th1 Cytokines

ABSTRACT During visceral leishmaniasis, the control of hepatic parasite burden is mainly due to granuloma assembly in a microenvironment consisting of both Th1 and Th2 components. Using enzyme-linked immunosorbent assay (ELISA) dosages, quantitative PCR (qPCR), immunohistochemistry, and flow cytometry, we studied the role of interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, during visceral leishmaniasis. We showed that a higher level of IL-33 was detected in the serum of patients with visceral leishmaniasis than in that from healthy donors and demonstrated the presence of IL-33+ cells in a liver biopsy specimen from a patient. Similarly, in BALB/c mice experimentally infected with L. donovani, a higher level of IL-33 was detected in the serum, as well as the presence of IL-33+ cells and ST2+ cells in the mouse liver. In ST2−/− BALB/c mice, better control of the hepatic parasite burden and reduced hepatomegaly were observed. This was associated with strong induction of Th1 cytokines (gamma interferon [IFN-γ] and IL-12) compared to the level in wild-type (WT) mice and better recruitment of myeloid cells associated with strongly induced chemokines (CCL2 and CXCL2) and receptors (CCR2 and CXCR2). Conversely, BALB/c mice treated twice weekly with recombinant IL-33 showed a dramatically reduced induction of Th1 cytokines and delayed inhibition of monocyte and neutrophil recruitment in the liver, which was associated with reduced KC/CXCL1 and CXCR2 expression. Taken together, our results suggest that IL-33 could be a new deleterious regulator of the hepatic immune response against Leishmania donovani, via the repression of the Th1 response and myeloid cell recruitment. IMPORTANCE Visceral leishmaniasis is a life-threatening systemic disease due to the Leishmania protozoa L. infantum and L. donovani and is ranked by the World Health Organization as the second most important protozoan parasitic disease after malaria for its grave morbidity, high mortality, and global distribution. Leishmania parasites subvert the host’s immune response to propagate to target organs, including the spleen, the bone marrow, and the liver. Control of hepatic parasite burdens depends on a delicate and poorly understood Th1/Th2 immune balance. To better understand this complex immune response, new cytokines are interesting targets for research studies. IL-33 is a newly described cytokine usually associated with Th2 response and involved in different diseases, including infectious diseases and hepatitis. Our results suggest that IL-33 could be a new factor of susceptibility and a potential prognostic marker during visceral leishmaniasis.

scholarly journals Cross-Protective Efficacy of a Prophylactic Leishmania donovani DNA Vaccine against Visceral and Cutaneous Murine Leishmaniasis

2005 ◽  
Vol 73 (2) ◽  
pp. 812-819 ◽  
Author(s):  
Ingrid Aguilar-Be ◽  
Renata da Silva Zardo ◽  
Edilma Paraguai de Souza ◽  
Gulnara Patrícia Borja-Cabrera ◽  
Miguel Rosado-Vallado ◽  
...  
Keyword(s):  
Immune Response ◽  
Dna Vaccine ◽  
Leishmania Donovani ◽  
Protective Efficacy ◽  
Humoral Response ◽  
Parasite Load ◽  
Lesion Size ◽  
Nucleoside Hydrolase ◽  
Main Component ◽  
And Control

ABSTRACT The fucose-mannose ligand (FML) complex of Leishmania donovani is a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovani and L. mexicana antigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovani antigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasi infection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicana infection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasi parasite load and a 65% reduction in L. mexicana lesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4+ T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmania species.

scholarly journals Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

2020 ◽  
Author(s):  
Subir Karmakar ◽  
Nevien Ismail ◽  
Fabiano Oliveira ◽  
James Oristian ◽  
Wen Wei Zhang ◽  
...  
Keyword(s):  
Immune Response ◽  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Leishmania Major ◽  
Protective Efficacy ◽  
Sand Fly ◽  
Endemic Region ◽  
Cytokines And Chemokines ◽  
Human Leishmaniasis ◽  
Strong Candidate

AbstractVisceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen−/−). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen−/− against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen−/− did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen−/−-immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen−/− immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-γ and TNF-α, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen−/− which showed equal protection as laboratory-grade LmCen−/− parasites in hamsters. Importantly, GLP-grade LmCen−/− parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

scholarly journals The Sex Differences of Morphology and Immunology of SIRS of Newborn Wistar Rats

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
A. M. Kosyreva
Keyword(s):  
Immune Response ◽  
Sex Differences ◽  
Wistar Rats ◽  
Inflammatory Diseases ◽  
Reproductive Age ◽  
Control Group ◽  
High Dose ◽  
Spleen Cells ◽  
Male And Female ◽  
Males And Females

The sex differences of infection and inflammatory diseases particularly appear at reproductive age and depend on the sex hormone level, varied between male and female. There are a few sets of data about the sex differences of infection and inflammatory diseases course, including systemic inflammatory response syndrome (SIRS) and sepsis, of newborns. The aim of our research was the estimation of morphological and immunological manifestation of SIRS of the newborn Wistar rats. Investigations were carried out on male and female two-day-old Wistar rats (10–12 g). SIRS was modeled by intraperitoneal injection of LPS (E. coli, O26: B6 strain, Sigma) in high dose—15 mg/kg. We did not find out any sex differences of the liver lesions severity between newborn males and females after LPS injection. The levels of endotoxin and estradiol in the serum, as the number of neutrophils in the intra-alveolar septa of the lungs, were higher in males than females with SIRS. Production of IL-2 and TNF-α by the spleen cells of males was higher than that in control group that reflects polarization predominantly on the Th1-type immune response. The secretion of IL-2, TNF-α, and IFN-γ by ConA activated spleen cells of females decreased that reflects the suppression of Th1-type immune response. We suppose that the LPS administration in the high dose causes the multidirectional reaction of the immune system of neonatal males and females Wistar rats.

Lupeol induces immunity and protective efficacy in a murine model against visceral leishmaniasis

Parasitology ◽  
2019 ◽  
Vol 146 (11) ◽  
pp. 1440-1450 ◽  
Author(s):  
Gurpreet Kaur ◽  
Kalpana Chauhan ◽  
Sukhbir Kaur
Keyword(s):  
Nitric Oxide ◽  
Immune Response ◽  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Protective Efficacy ◽  
Parasite Burden ◽  
Delayed Type Hypersensitivity ◽  
Reference Drug ◽  
Th2 Immune Response

AbstractThe available chemotherapeutics for the cure of visceral leishmaniasis (VL) are linked with many detrimental effects. Moreover, VL is associated with the suppression of protective Th1 immune response of the host and induction of disease exaggerating Th2 immune response. Therefore, there is an urgent requirement of therapeutics which can augment the immune status of the host to cure this disease. In the current investigation, the antileishmanial potential of lupeol was monitored in vitro and in vivo in inbred BALB/c mice against Leishmania donovani. Lupeol showed potent antipromastigote activity via arresting parasites at sub G0/G1 phase in vitro. Lupeol significantly decreased the splenic parasite burden by inducing strong delayed-type hypersensitivity responses in contrary to untreated infected animals. The therapeutic efficacy of lupeol was observed to be similar to the reference drug, AmB. Treatment of infected animals with lupeol depicted enhanced levels of T cells and Th1 cytokines in contrast to only infected controls. Further lupeol treatment upregulated the levels of nuclear factor κ B and nitric oxide synthase genes and elevated the production of reactive oxygen species and nitric oxide. Unlike AmB, lupeol-treated infected animals did not show any toxicity. These findings are promising and indicate that lupeol can serve as a prototype drug for the cure of VL.

scholarly journals Evaluation of the Efficacy of Outer Membrane Protein 31 Vaccine Formulations for Protection against Brucella canis in BALB/c Mice

2014 ◽  
Vol 21 (12) ◽  
pp. 1689-1694 ◽  
Author(s):  
Maria Clausse ◽  
Alejandra G. Díaz ◽  
Andrés E. Ibañez ◽  
Juliana Cassataro ◽  
Guillermo H. Giambartolomei ◽  
...  
Keyword(s):  
Preventive Measures ◽  
Protective Efficacy ◽  
Interleukin 4 ◽  
Spleen Cells ◽  
Th2 Response ◽  
Content Type ◽  
Control Programs ◽  
Brucella Canis ◽  
Canine Brucellosis

ABSTRACTCanine brucellosis is an infectious disease caused by the Gram-negative bacteriumBrucella canis. Unlike conventional control programs for other species of the genusBrucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinantBrucellaOmp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy againstB. canisinfection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) afterin vitrostimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection againstB. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine againstB. canisinfection.

Safety and Immunogenicity of High Dose Trivalent Inactivated Influenza Vaccine in Pediatric Patients with Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3531-3531
Author(s):  
Meghann Pine McManus ◽  
Haydar Frangoul ◽  
Jonathan McCullers ◽  
Wenli Wang ◽  
Steve Ampath ◽  
...  
Keyword(s):  
Immune Response ◽  
Acute Lymphoblastic Leukemia ◽  
Influenza Vaccine ◽  
Pediatric Patients ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Vaccine Antigen ◽  
High Dose ◽  
Children With Cancer ◽  
Significant Difference

Abstract Abstract 3531 Background: Influenza is an important cause of morbidity and mortality worldwide. Most deaths outside the elderly population are seen in other high risk groups, such as immunocompromised individuals. Compared to the general population, children with cancer have a higher frequency of influenza infections, have symptoms lasting twice as long and are more likely to require hospitalization, all of which may lead to delays in their chemotherapy. It is recommended that children with cancer receive a yearly trivalent influenza vaccine (TIV) and studies show that children with acute lymphoblastic leukemia (ALL) do mount an immune response to the TIV, although they often have lower titers and seroresponse rates compared to healthy controls. Recently, a high dose (HD) TIV was found to provide a statistically significant increase in the level of antibody response in elderly patients compared to the standard dose (SD) TIV. We hypothesized that the HD TIV would be well tolerated and more immunogenic compared to the SD TIV in pediatric patients with ALL. Methods: This was a randomized, double-blind, phase I safety and immunogenicity trial comparing HD to SD TIV in children with ALL aged 3–17 years, at least one month into chemotherapy and in 1stcomplete remission. Subjects were randomized 2:1 to receive either 0.5mL of HD (60ug per antigen) or SD (15ug per antigen) 2010–11 or 2011–12 TIV. Local and systemic reactions were collected for 7 days after each vaccination. HAI titers to influenza virus antigens as well as complete blood count, quantitative CD4, CD8, CD19 and serum IgG levels were measured before and 28–35 days after vaccination. In year 1, no blood was drawn before dose 2 if a second dose was required. Results: 50 subjects were enrolled (20 in year 1, 30 in year 2). Mean age was 8.25 years (range 4.7 – 12.3 years) and 62% were male. The majority of patients (78%) were in the maintenance phase of therapy. 34 subjects received the HD TIV and 16 subjects received the SD TIV (mean age 7.8 vs. 9.3 years), with 11 subjects receiving 2 doses (9 in HD and 2 in SD groups). The only significant difference noted between the HD and SD TIV group was mean total CD19 count (29 vs. 56, p=0.027). There were no significant differences reported in local or systemic symptoms, except fatigue/malaise and headaches were reported more frequently in the SD TIV group (p=0.008 and p=0.03, respectively). No severe adverse events were attributed to vaccination. The immune response measured by a ≥ 4-fold rise in titers for each vaccine antigen were similar in both the HD and SD TIV groups after 1 or 2 vaccines, respectively (A/California: p=0.12, p=0.46; A/Perth: p=0.35, p=0.34; B/Brisbane: p=0.42, p=0.89). Please see refer to tables 1 and 2 for further immunogenicity results. Conclusion: No differences were noted between the HD and SD TIV groups for solicited systemic and local reactions. The immune response appeared similar in both vaccine groups. A phase 3 trial is planned to determine the immunogenicity of HD versus SD TIV in the pediatric ALL population. Disclosures: No relevant conflicts of interest to declare.

HIV-1 Accessory Proteins: Which one is Potentially Effective in Diagnosis and Vaccine Development?

2020 ◽  
Vol 28 ◽  
Author(s):  
Alireza Milani ◽  
Kazem Baesi ◽  
Elnaz Agi ◽  
Ghazal Marouf ◽  
Maryam Ahmadi ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Development ◽  
Treated Group ◽  
Vaccine Antigen ◽  
Accessory Proteins ◽  
Serological Method ◽  
Th2 Immune Response ◽  
Untreated Individuals ◽  
Immunogenic Properties ◽  
Hiv 1

Background:: The combination antiretroviral therapy (cART) could increase the number of circulating naive CD4 T lymphocytes, but was not able to eradicate human immunodeficiency virus-1 (HIV-1) infection. Objective:: Thus, induction of strong immune responses is important for control of HIV-1 infection. Furthermore, a simple and perfect serological method is required to detect virus in untreated-, treated- and drug resistant- HIV-1 infected individuals. Methods:: This study was conducted to assess and compare immunogenic properties of Nef, Vif, Vpr and Vpu accessory proteins as an antigen candidate in mice and their diagnostic importance in human as a biomarker. Results:: Our data showed that in mice, all heterologous prime/ boost regimens were more potent than homologous prime/ boost regimens in eliciting Th1 response and Granzyme B secretion as CTL activity. Moreover, the Nef, Vpu and Vif proteins could significantly increase Th1 immune response. In contrast, the Vpr protein could considerably induce Th2 immune response. On the other hand, among four accessory proteins, HIV-1 Vpu could significantly detect treated group from untreated group as a possible biomarker in human. Conclusion:: Generally, among accessory proteins, Nef, Vpu and Vif antigens were potentially more suitable vaccine antigen candidates than Vpr antigen. Human antibodies against all these proteins were higher in HIV-1 different groups than healthy group. Among them, Vpu was known as a potent antigen in diagnosis of treated from untreated individuals. The potency of accessory proteins as an antigen candidate in an animal model and a human cohort study are underway.

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