Juana Calderon-Amador

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

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.

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

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.

Mycobacterial Strains of Different Virulence Trigger Dissimilar Patterns of Immune System Activation In Vivo

Tuberculosis (TB), one of the major world health problems, is a chronic infection caused by members of the Mycobacterium tuberculosis complex (MTC). In 2009, tuberculosis (TB) caused 1.7 million deaths and 9.4 million new cases. Although recent efforts to improve TB prevention, diagnosis and treatment have contributed to a 35% decrease in the death rate, the emergence of mycobacterial strains with highly virulent phenotypes combined with pandemic HIV infections has added new challenges to control TB.

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

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 armadillos 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 animals 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 armadillos LC will incite studies of leprosy and immunity in this animal model.

Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c+ putative dendritic cells in horse lymph nodes.

A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in peripheral blood cell suspensions and in cryosections of horse lymph nodes. Only the serum against peptide 2 was capable of identifying cells in suspension and in situ by immunohistochemistry, some with evident dendritic morphology. Using this approach, an immunogenic epitope exposed in CD11c was identified in cells from horse lymph node in situ.

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

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.

Neonate Antigen Presenting Cells within Murine Intestinal Muscular Layer

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.