Publications by authors named "Juana Calderón-Amador"

17 Publications

  • Page 1 of 1

Langerhans Cells From Mice at Birth Express Endocytic- and Pattern Recognition-Receptors, Migrate to Draining Lymph Nodes Ferrying Antigen and Activate Neonatal T Cells .

Front Immunol 2020 27;11:744. Epub 2020 Apr 27.

Department of Cell Biology, Center for Advanced Research, The National Polytechnic Institute, Cinvestav-IPN, Mexico City, Mexico.

Antigen capturing at the periphery is one of the earliest, crucial functions of antigen-presenting cells (APCs) to initiate immune responses. Langerhans cells (LCs), the epidermal APCs migrate to draining lymph nodes (DLNs) upon acquiring antigens. An arsenal of endocytic molecules is available to this end, including lectins and pathogen recognition receptors (PRRs). However, cutaneous LCs are poorly defined in the early neonatal period. We assessed endocytic molecules expression : Mannose (CD206)-, Scavenger (SRA/CD204)-, Complement (CD2l, CDllb)-, and Fc-Receptors (CD16/32, CD23) as well as CD1d, CD14, CD205, Langerin (CD207), MHCII, and TLR4 in unperturbed epidermal LCs from both adult and early neonatal mice. As most of these markers were negative at birth (day 0), LC presence was revealed with the conspicuous, epidermal LC-restricted ADPase (and confirmed with CD45) staining detecting that they were as numerous as adult ones. Unexpectedly, most LCs at day 0 expressed CD14 and CD204 while very few were MHCII+ and TLR4+. In contrast, adult LCs lacked all these markers except Langerin, CD205, CD11b, MHCII and TLR4. Intriguingly, the CD204+ and CD14+ LCs predominant at day 0, apparently disappeared by day 4. Upon cutaneous FITC application, LCs were reduced in the skin and a CD204+MHCII+FITC+ population with high levels of CD86 subsequently appeared in DLNs, with a concomitant increased percentage of CD3+CD69+ T cells, strongly suggesting that neonatal LCs were able both to ferry the cutaneous antigen into DLNs and to activate neonatal T cells . Cell cycle analysis indicated that neonatal T cells in DLNs responded with proliferation. Our study reveals that epidermal LCs are present at birth, but their repertoire of endocytic molecules and PRRs differs to that of adult ones. We believe this to be the first description of CDl4, CD204 and TLR4 in neonatal epidermal LCs . Newborns' LCs express molecules to detect antigens during early postnatal periods, are able to take up local antigens and to ferry them into DLNs conveying the information to responsive neonatal T cells.
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http://dx.doi.org/10.3389/fimmu.2020.00744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197463PMC
March 2021

Proteomic Analysis of Membrane Blebs of 2308 and RB51 and Their Evaluation as an Acellular Vaccine.

Front Microbiol 2019 29;10:2714. Epub 2019 Nov 29.

Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico.

Membrane blebs are released from Gram-negative bacteria, however, little is known about blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and analysis. The second aim was to evaluate the use of membrane blebs of 2308 and RB51 as an acellular vaccine and . To achieve these aims, membrane blebs from 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. membrane blebs were used as a "vaccine" to induce an immune response in BALB/c mice, using the strain RB51 as a positive vaccine control. After subsequent challenge with 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth induced similar protective immune responses as well as the vaccine RB51 after the challenge with virulent strain 2308 ( < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19CD69) Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.
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http://dx.doi.org/10.3389/fmicb.2019.02714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895012PMC
November 2019

In vivo anti-arthritic effect and repeated dose toxicity of standardized methanolic extracts of Buddleja cordata Kunth (Scrophulariaceae) wild plant leaves and cell culture.

J Ethnopharmacol 2019 Aug 26;240:111875. Epub 2019 Apr 26.

Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa (UAM-I), Av. San Rafael Atlixco 186, Colonia Vicentina, Delegación Iztapalapa, 09340, Ciudad de México (CDMX), Mexico. Electronic address:

Ethnopharmacological Relevance: Buddleja cordata Humb. Bonpl. & Kunth, known by the population as Tepozán blanco, is a shrub plant used in traditional herbal medicine in Mexico for the treatment of tumors, cancer, sores, skin burns, rheumatic pains and diseases related to inflammatory processes such as arthritis; authors adjudicate this etno-medicinal effect to the presence of secondary metabolites in the plant such as verbascoside, however due to its low concentration in recent years biotechnological tools are applied as cell culture to biosynthesize these pharmacological active metabolites in greater quantities.

Aim Of The Study: Evaluate the possible toxic effect after a daily administration of MeOH extracts from wild plant leaves (Bc-Wp), and cell culture (Bc-Cc) of B. cordata for 28 days, and after their anti-edematous and antioxidant activities in vivo, as well their effect on the cytokines profile during experimental arthritis induced by complete Freund's adjuvant.

Materials And Methods: Both extracts were evaluated in CD1 male mice first in a toxicity test of repeated dose administrations (1 g/kg) for 28 days, after which pharmacological activity of both extracts was measure during experimental induced arthritis where three doses were tested, at the end of the study edema formation, body weight gain and antioxidant activity were measure in edema and ganglionic tissues. Finally, dose that exerted the best protective effect (250 mg/kg) was evaluated to quantify its effect over ganglionic tissue concentration of lymphocytes T CD4, and cytokines (IL-1β, TNF-α and IL-10), as well histological analysis in arthritic mice.

Results: Both extracts (Bc-Wp and Bc-Cc) did not exert lethality, however body weight gain and food in-take were lower than in healthy mice administered with vehicles, also extract-treated animals showed a decrease in serum lipid concentration and only Bc-Wp extract treated animals decrease serum alkaline phosphatase after 28 daily administration compared to healthy un-treated mice. During experimental arthritis best inhibition effect over edema development was observed in those animals administered with both extracts at dose of 250 mg/kg (Bc-Wp and Bc-Cc) on day 28, compared to CFA un-treated mice. Also both extracts reduce oxidative damage over lipids and proteins at the same dose, in both ganglionic and edema tissue, as well antioxidant enzymatic response was reduced in both tissues compared to arthritic un-treated group. In ganglionic tissue of arthritic mice, CD4 lymphocytes concentration was reduced by Bc-Wp and Bc-Cc treatment (250 mg/kg) respectively, as well IL-1β, and TNF-α levels. Only arthritic animals treated with Bc-Cc extract at 250 mg/kg generated a significant increase of IL-10 doubling the levels compared to CFA un-treated group. Histological analysis of popliteal ganglion showed that both extracts decrease the incidence of lytic lesions, lipid inclusions and leukocyte infiltration.

Conclusion: Methanolic extracts of wild Buddleja cordata and its cell cultures did not generated lethality after a daily administration for 28 days at 1 g/kg, but it was observed that both showed a lipid-lowering effect. Also at dose of 250 mg/kg both extracts exerted anti-edematous, protection against the oxidation of lipid and proteins, regulation on antioxidant enzymatic response, down-regulation on lymphocytes CD4 producers of IL-1β and TNF-α, an increase in IL-10 levels, which caused a decrease in leukocyte infiltration in ganglionic tissue during experimental arthritis.
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http://dx.doi.org/10.1016/j.jep.2019.111875DOI Listing
August 2019

The Outer Membrane Vesicles of ATCC 7966: A Proteomic Analysis and Effect on Host Cells.

Front Microbiol 2018 16;9:2765. Epub 2018 Nov 16.

Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico.

Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of OMVs is missing. In this study we analyzed the composition of purified OMVs from ATCC 7966 by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90-170 nm. Moreover, 211 unique proteins were found in OMVs from ; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from ATCC 7966 induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of ATCC 7966 and their interaction with the host cell.
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http://dx.doi.org/10.3389/fmicb.2018.02765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250952PMC
November 2018

Corrigendum: Anti-Lipid IgG Antibodies Are Produced Germinal Centers in a Murine Model Resembling Human Lupus.

Front Immunol 2017 12;8:440. Epub 2017 Apr 12.

Department of Cell Biology, Center for Research and Advanced Studies, CINVESTAV-IPN, Instituto Politécnico Nacional (IPN) , Mexico City , Mexico.

[This corrects the article on p. 396 in vol. 7, PMID: 27746783.].
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http://dx.doi.org/10.3389/fimmu.2017.00440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388691PMC
April 2017

Immunization of Newborn Mice Accelerates the Architectural Maturation of Lymph Nodes, But AID-Dependent IgG Responses Are Still Delayed Compared to the Adult.

Front Immunol 2017 19;8:13. Epub 2017 Jan 19.

Department of Cell Biology, Center for Advanced Research, The National Polytechnic Institute, Cinvestav-IPN , Mexico City , Mexico.

Lymph nodes (LNs) have evolved to maximize antigen (Ag) collection and presentation as well as lymphocyte proliferation and differentiation-processes that are spatially regulated by stromal cell subsets, including fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs). Here, we showed that naïve neonatal mice have poorly organized LNs with few B and T cells and undetectable FDCs, whereas adult LNs have numerous B cells and large FDC networks. Interestingly, immunization on the day of birth accelerated B cell accumulation and T cell recruitment into follicles as well as FDC maturation and FRC organization in neonatal LNs. However, compared to adults, the formation of germinal centers was both delayed and reduced following immunization of neonatal mice. Although immunized neonates poorly expressed activation-induced cytidine deaminase (AID), they were able to produce Ag-specific IgGs, but with lower titers than adults. Interestingly, the Ag-specific IgM response in neonates was similar to that in adults. These results suggest that despite an accelerated structural maturation of LNs in neonates following vaccination, the B cell response is still delayed and reduced in its ability to isotype switch most likely due to poor AID expression. Of note, naïve pups born to Ag-immunized mothers had high titers of Ag-specific IgGs from day 0 (at birth). These transferred antibodies confirm a mother-derived coverage to neonates for Ags to which mothers (and most likely neonates) are exposed, thus protecting the neonates while they produce their own antibodies. Finally, the type of Ag used in this study and the results obtained also indicate that T cell help would be operating at this stage of life. Thus, neonatal immune system might not be intrinsically immature but rather evolutionary adapted to cope with Ags at birth.
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http://dx.doi.org/10.3389/fimmu.2017.00013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5243854PMC
January 2017

Anti-Lipid IgG Antibodies Are Produced Germinal Centers in a Murine Model Resembling Human Lupus.

Front Immunol 2016 29;7:396. Epub 2016 Sep 29.

Department of Cell Biology, Center for Research and Advanced Studies, CINVESTAV-IPN, National Polytechnic Institute , Mexico City , Mexico.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1, CD19, CD138) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD, CD19, PNA) specific for NPA in the draining lymph nodes and the spleen, and we identified the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220, Blimp1) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.
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http://dx.doi.org/10.3389/fimmu.2016.00396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040728PMC
September 2016

Germinal center reaction following cutaneous dengue virus infection in immune-competent mice.

Front Immunol 2015 24;6:188. Epub 2015 Apr 24.

Department of Cell Biology, Center for Advanced Research, National Polytechnic Institute, Cinvestav-IPN , Mexico City , Mexico.

Dengue virus (DENV) has four serotypes, which can cause from asymptomatic disease to severe dengue. Heterologous secondary infections have been associated to a greater risk of potentially fatal dengue due to non-neutralizing memory antibodies (Abs), which facilitate the infection, such as anti-precursor membrane (prM) Abs, among other mechanisms. Usually, class-switched memory Abs are generated mainly through germinal centers (GCs). However, the cellular events underlying these Ab responses to DENV, especially during repeated/secondary infections, have been poorly studied. We wanted to know whether there is involvement of GC reactions during cutaneous DENV infection and whether there is any sort of preferential Ab responses to defined viral proteins. Intradermal DENV inoculation at a relatively low dose efficiently infects immune-competent BALB/c mice, inducing higher quantities of DENV-specific GC B cells and larger GCs than the control conditions. Interestingly, GCs exhibited as much prM as envelope (E) and non-structural 3 viral proteins in situ. Intriguingly, despite the much larger abundance of E protein than of prM protein in the virions, infected animals showed similar amounts of circulating Abs and Ag-specific GC B cells both for prM and for E proteins, even significantly higher for prM. To the best of our knowledge, there are no reports of the GC responses during DENV infection. This relatively stronger anti-prM response could be triggered by DENV to preferentially promote Abs against certain viral proteins, which might favor infections by facilitating DENV invasion of host cells. It is thus conceivably that DENV might have evolved to induce this kind of Ab responses.
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http://dx.doi.org/10.3389/fimmu.2015.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408864PMC
May 2015

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

PLoS One 2015 27;10(4):e0124828. Epub 2015 Apr 27.

Department of Cell Biology, Cinvestav-IPN, Ciudad de México, Mexico.

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411092PMC
February 2016

Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells.

Springerplus 2015 2;4:161. Epub 2015 Apr 2.

Department of Cell Biology, Cinvestav-IPN. AV. IPN No 2508, Zacatenco, C.P. 07330 D.F México.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this "late" induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.
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http://dx.doi.org/10.1186/s40064-015-0932-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392041PMC
April 2015

Novel monoclonal antibody against alphaX subunit from horse CD11c/CD18 integrin.

Vet Immunol Immunopathol 2015 Apr 12;164(3-4):220-6. Epub 2015 Feb 12.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia - Universidad Nacional Autonoma de Mexico, Avenida Universidad, 2001, Colonia Chamilpa, Apartado Postal 510-3, Cuernavaca, Morelos, Mexico. Electronic address:

The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph node tissue sections, mAb-1C4 marked cells in situ, some with apparent dendritic morphology. Thus the mAb generated to a recombinant epitope from horse CD11c identified the molecule in intact cells within horse lymphoid tissue. By the labelling intensity, the histological location (paracortical and interfollicular areas) and the apparent morphology of the marked cells, we can say that these are putative horse dendritic cells (DCs). The development of a mAb to horse CD11c provides a new tool to better study the horse DC biology and opens other biotechnological avenues, such as DC targeting-based vaccines.
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http://dx.doi.org/10.1016/j.vetimm.2015.02.002DOI Listing
April 2015

Neonate antigen presenting cells within murine intestinal muscular layer.

Immunol Invest 2012 23;41(1):104-16. Epub 2011 Jun 23.

Department of Cell Biology, Center for Advanced Research, CINVESTAV-IPN, Mexico City, Mexico.

The intestinal mucosa is exposed to a vast antigenic contact. Several antigen presenting cell (APCs) have been described within the gut associated lymphoid tissue (GALT) (Peyer's patches, lamina propria, mesenteric lymph nodes, muscular layer); however, this has been done almost exclusively in adult organisms. As there is no characterization of intestinal muscular layer's APCs during early neonate development we adapted the conventional technique used in adults, to the neonate intestine. We obtained the intestinal muscular layer from early neonates (days 0-3 upon birth) and from young mice (2 and 3 weeks after birth). A planar network of CD45(+), MHC-II(+), DEC-205(+) cells with irregular, some with prominent dendritic morphology was found at birth under basal physiological conditions, whereas Langerin(+) DCs appeared after two weeks. The variations seen in CD45(+), MHC-II(+) and DEC-205(+) cells along the early neonatal development, could be related to the new challenges by intestinal antigen exposure from the newborn diet (breast milk, solid food), and to important environmental changes (start walking, exploring the surroundings, etc). Our study reveals the presence of APCs in intestinal muscular layer at birth, and their subsequent changes in physiological, non-induced conditions, contributing basic information about these cells in the neonate intestinal immune system.
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http://dx.doi.org/10.3109/08820139.2011.586394DOI Listing
March 2012

Characterization of langerhans cells in epidermal sheets along the body of Armadillo (Dasypus novemcinctus).

Vet Immunol Immunopathol 2008 Aug 1;124(3-4):220-9. Epub 2008 Apr 1.

Department of Immunology, National School of Biological Sciences ENCB-IPN, Mexico City, Mexico.

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.
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http://dx.doi.org/10.1016/j.vetimm.2008.03.011DOI Listing
August 2008

Network of dendritic cells within the muscular layer of the mouse intestine.

Proc Natl Acad Sci U S A 2005 Dec 16;102(52):19039-44. Epub 2005 Dec 16.

Department of Immunology, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala, Casco de Santo Tomás, 11340, Mexico.

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcgamma receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c(+) populations of CD45(+) CD205(+), CD19(-) leukocytes, MHC II(+) and MHC II(-). When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4(+) and CD8(+) T cell antigen receptor-transgenic T cells. In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine.
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http://dx.doi.org/10.1073/pnas.0504253102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1316057PMC
December 2005

Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells.

Int J Exp Pathol 2005 Oct;86(5):323-34

Department of Experimental Pathology, Center for Advanced Research, CINVESTAV-IPN, Mexico City, Mexico.

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.
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http://dx.doi.org/10.1111/j.0959-9673.2005.00445.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517443PMC
October 2005

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis.

Int J Exp Pathol 2004 Jun;85(3):135-45

Department of Experimental Pathology, Center for Advanced Research CINVESTAV-IPN, Mexico City, Mexico.

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl-Neelsen staining. CD11c(+) DC and CD14(+) cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII(+) cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14-21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24-48 h). Regarding granuloma constitution, highly bacillary CD14(+) and SRA(+) cells were centrally located. MHC-CII(+) cells were more peripheral, with less mycobacteria. CD11c(+) cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl-Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth.
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http://dx.doi.org/10.1111/j.0959-9673.2004.00381.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517470PMC
June 2004