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To ascertain the in vivo role of mycobacterial lipids phthiocerol dimycocerosates (PDIM) in experimental murine tuberculosis (Tb), airways infection was used to compare the parental virulent clinical isolate MT103 with its mutant fadD26, lacking PDIM. Lungs were assessed as the Tb-target organ and mediastinal lymph nodes as the corresponding lymphoid tissue, in order to quantify: the major T-cell subsets (CD4+/CD8+/gammadelta+) and their activation kinetics, bacillary burden, and in vivo cytotoxicity against inoculated target cells loaded with mycobacterial Ags. After 4 weeks, infection augmented total and activated CD4+ and CD8+ T cells in lungs and nodes mainly with MT103, while gammadelta+ T cells increased earlier in nodes. MT103 bacillary burden was bigger and appeared earlier than the mutant fadD26, especially in the lung than in mediastinal nodes. At day 14 of MT103 infection, there was no cytotoxicity in lungs and nodes; while with fadD26 there was some in the nodes. At day 21 of MT103 infection, important cytotoxicity was detected only in lungs; while with fadD26 both tissues showed important activity. Interestingly, unlike the infection with fadD26, cytotoxicity under MT103 fell considerably in the target organ (lung) from days 21 to 60, the advanced phase. Although upon airways infection both mycobacteria behaved similarly regarding T cell (CD4/CD8/gammadelta) stimulation kinetics; they differed in the magnitude of these responses, in the bacterial load within tissues, and to trigger in vivo cytotoxicity in lungs and regional lymph nodes. This highlights the relevance of certain mycobacterial lipids to modify crucial effector branches of immunity.

Dendritic cells (DC) are often arranged in planar layers in tissues with high antigenic exposure, such as skin and mucosae. Providing an en face view, this arrangement optimizes in situ analysis regarding morphology (even of individual dendrites), topographic distribution (regular/clustered) and quantification. The few reports on human genital DC usually utilize single markers and conventional sections, restricting immunolabelling only to cell parts sectioned by the cut. To better assess DC in situ, we labelled epithelial sheets, prepared from fresh cervix biopsies, with antibodies to major histocompatibility complex (MHC)-CII, CD1a and Langerin, revealing (with each of these markers) a dense DC network in a planar-like, regular distribution. Using the hybrid capture system to detect the high-risk mucotropic human papilloma virus (HPV) group, 16 positive and five negative women were studied and the results were compared between these groups. DC frequency per area was substantially reduced (to approximately 50% for the three markers) in samples from all HPV-infected patients compared with samples from controls. Unlike HPV(-) samples, Langerin(+) DC in HPV(+) cervix exhibited a highly accentuated dendritic appearance. We believe this to be the first study using these three DC-restricted markers (Langerin, CD1a and MHC-CII) in cervical epithelial sheets from high-risk HPV(+) donors and also the first study to demonstrate the morphological and quantitative changes triggered by high-risk HPV infection. Cervical DC reduction in early, premalignant high-risk HPV infection might represent viral subversion strategies interfering with efficient antigen handling by the immune system's peripheral sentinels, the DC, perhaps hampering appropriate recruitment and subsequent development of effector (cytotoxic) T cells.

Dengue virus (DV) infections are serious causes of morbidity and mortality worldwide, and adaptive (secondary) immune response appears to influence the severity of this arboviral disease (Morens 1994; Halstead & O'Rourke 1977; Rigau-Perez et al. 1998). There are an estimated 50–100 million cases of dengue fever and 250,000-500,000 cases of dengue haemorrhagic fever (DHF) annually in the world (WHO 1997). The dengue shock syndrome (DSS), the most severe and potentially fatal form of the disease, especially in developing countries, is less frequent. In the model proposed here, we have chosen to examine DV2, because it is the prevalent serotype (Gomez-Dantes et al. 2004). The high seroprevalence together with the co-circulation of multiple serotypes indicates that Mexico could be in risk of an important DHF outbreak (Gomez-Dantes et al. 2004). The exact pathogenic mechanisms following DV infection are not well understood, especially in the severe forms of the infection (DHF and DSS). The immune response to DV seems at least partially involved in the process (Halstead 1988; Avirutnan et al. 1998; Diamond et al. 2000). Several theories have been advanced including facilitation of DV infection by non-neutralizing antibodies (Halstead & O'Rourke 1977; Halstead et al. 1977; Morens 1994), the potential differential virulence of each infecting serotype (Leitmeyer et al. 1999; Kawaguchi et al. 2003), CD4-CD8 T-cell cross-reactivity and host factors such as a particular, but yet undefined susceptibility (Klenerman & Zinkernagel 1998; Mongkolsapaya et al. 2003). While there is an excellent research on dengue about the molecular structure and epidemiology of this virus, the basic mechanisms of the immunopathology still remain obscure (Haywood 1994; Kuhn et al. 2002). For instance, until very recently (Johnston et al. 2000; Wu et al. 2000), it was unknown whether DV was able to replicate in cutaneous cells. Likewise, there are two other features largely unexplored regarding DV infection: (i) the very early stages of the infection, because, by necessity, patients are examined when disease is almost over, when the patients are under recovery or when they have entered into DSS and (ii) which type of responses, if any, occur cutaneously, in situ, following DV inoculation when mosquitoes feed. We wanted to develop an in situ approach that would enable us to study the early immune pathology of the cellular events regarding the infectious process and the first reactions of the peripheral immune system, the dendritic cells (DCs). DCs constitute a system of highly dynamic sentinel cells, whose most studied element in peripheral non-lymphoid tissues is the epidermal Langerhans cells (LCs). It is now known that LCs perform multiple tasks including antigen capturing and processing, antigen ferrying to regional lymphoid tissues, and ultimately, cognate antigen presentation to naive lymphocytes, once in secondary lymphoid organs (Flores-Romo 2001). Under the influence of a variety of stimuli such as cytokines, antigens or microbial products, LCs become activated, start to migrate into the dermis and concomitantly to up-regulate antigen-presenting molecules and to express co-stimulatory and maturation markers (Cumberbatch & Kimber 1992; Cumberbatch et al. 1997). Nevertheless, despite that LCs as sensors of the external antigenic world are the most exposed cells of the immune system during mosquito feeding, and the initial virus inoculation through skin, the interactions of these cells with DV and the outcome of such potential interplay regarding both the virus and the LC have received little attention in situ (Wu et al. 2000). We attempted to address some of these issues by establishing a model with fresh, healthy, non-cadaveric human skin explants exposed to DV, to analyse whether local infection and viral replication are feasible experimentally, to assess the most early phases of the initiation of the immune responses in situ and the immediate local repercussions of viral inoculation upon local antigen-presenting cells (APCs), particularly on the DCs, the sentinel posts of the immune system in the skin.

Pulmonary tuberculosis, an infection caused by Mycobacterium tuberculosis has been declared a global health emergency by the World Health Organization (WHO). Tuberculosis is a re-emergent expanding disease, responsible for a great mortality and morbidity throughout the world. It has been estimated that one-third of the world's population is currently infected with M. tuberculosis, with 8 million new cases per year. Nevertheless, only 5–10% will develop the chronic pulmonary disease (Dye et al. 1999; WHO 2000). Most people will be able to control the infection through efficient immune responses, especially the cellular arm of immunity. However, the bacilli might not be eliminated completely and may persist dormant for years in a latent state, a phenomenon not well understood yet (Flynn & Ernest 2000; Hernandez-Pando et al. 2000; Manabe & Bishai 2000). M. tuberculosis spreads from a person with pulmonary tuberculosis through aerosols, entering the body via the respiratory tract, reaching the lungs where phagocytic cells become infected, especially the alveolar macrophages (AMs). Apparently, M. tuberculosis can survive within AM for long-lasting periods, causing either a latent infection or subsequently developing the pulmonary disease (Wiegeshaus et al. 1989; Andersen 1997). This bacillus has evolved several mechanisms to evade the host immune responses, including the inhibition of antimicrobial activity of macrophages (Schluger & Rom 1998). It is known that the first line of defence against M. tuberculosis is the AM, which interacts with the bacilli through different surface molecules like complement, mannose and Toll-like receptors (Arthur & Dannenberg 1991; Schluger & Rom 1998). Despite the fact that dendritic cells (DCs) are present throughout the respiratory tract, from the upper nasal mucosa to parenchymal lung tissue, little is known about lung DCs in vivo during tuberculosis, either in the naturally occurring disease or in experimental models of infection. Lung DCs are found within pleura and the alveolar septal walls; additionally they can be found, although in very small numbers, in the alveolar spaces (Holt & Schon–Hegrad 1987; McWilliam et al. 1995; Holt 2000). In vitro assays have shown that monocyte-derived DCs and macrophages internalize the mycobacteria, resulting in DC maturation and activation with a differential production of cytokines. Interleukin-12 (IL-12) was almost exclusively produced by DCs, while macrophages produced IL-10 and the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-6 (Mohagheghpour et al. 2000; Giacomini et al. 2001). M. tuberculosis grew equally well within nonactivated DCs and macrophages; however, activation with IFN-γ and LPS inhibited the growth of this intracellular microorganism. While macrophages can kill the intracellular bacteria, DCs seem unable to do so and, apparently, only restrict the growth of this bacillus (Bodnar et al. 2001). Experiments conducted in vivo with the M. bovis (BCG) showed that, following intravenous administration, bacilli were detected in similar percentage in splenic macrophages and in splenic DCs. After 2 weeks, BCG bacilli were still viable without apparent growth within DCs (Jiao et al. 2001). In this study, using four different markers in situ, we describe the main lung antigen-presenting cells (APCs) in the susceptible BALB/c mice during experimental pulmonary tuberculosis, which is induced by intratracheal inoculation of the virulent strain of M. tuberculosis H37Rv. Pulmonary infection with M. tuberculosis induced an increase of parenchymal lung APC; these cells displayed differential tissue distribution and morphological features during the infection and, apparently, also a differential capacity to interact with bacilli in vivo.

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.

Background Actinic Prurigo (AP) is a chronic pruritic dermatosis of unknown cause affecting sun exposed skin in defined ethnic groups with characteristic MHC alleles. However, the cutaneous dendritic cells have not been assessed. Objective To assess in situ the epidermal Langerhans Cell (LC) status in Actinic Prurigo. Study design Fresh skin samples from three AP patients were used to evaluate in situ the epidermal LC, comparing lesional and non-lesional sites in each subject. Setting AP patients attending the Dermatology Department at the Hospital M. Gea-Gonzalez in Mexico city. Methods Lesional and non-lesional skin samples were taken from each subject to prepare both epidermal sheets and conventional tissue sections. Three markers restricted to LC in epidermis (CD1a, ATPase, MHC-II) were used to quantify the LC per area in epidermal sheets. Results Compared to non-lesional skin from the same subject, a significant reduction in the number of LC per area of epidermis was found in lesional skin; with any of the three markers evaluated. Conclusion The frequency of epidermal LC decreases importantly in lesional skin from AP patients.

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.