Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.     

Schistosoma mansoni: phospholipid methylation and the escape of schistosomula from in vitro cytotoxic reaction

The ability of Schistosoma mansoni schistosomula to evade in vitro cytotoxic activity of antibodies plus complement is shown to be increased by incubation with Concanavalin A (Con A) or with non-immune inactivated human serum. This effect was not observed if S-adenosyl-homocysteine (SAH) a methyltransferase inhibitor was added to the incubation medium. Methyl group incorporation occurs in schistosomulum phospholipids if parasites are incubated in Earle's balanced salt solution. This incorporation is increased by Con A addition and this increase is inhibited by SAH. Supernatants of schistosomula incubated in culture media containing Con A were able to promote phospholipid methylation, showing that methyltransferases were liberated into the culture media. The possible roles played by these phenomena in host-parasite interactions are discussed.     

Genetic predisposition to leprosy: A major gene reveals novel pathways of immunity to Mycobacterium leprae

The elucidation of the genetic control of susceptibility to common infectious diseases is expected to provide new and more effective tools for prevention and control of some of the most pressings health needs on a global scale. A major advantage of whole genome based genetic approaches is that no a priori assumptions about mechanisms of pathogenesis need to be made in these studies. Hence, genetic studies can identify previously unrecognized pathways of disease susceptibility and tag critical pathogenic events for further biochemical, immunological or physiological analysis. We have applied this strategy to leprosy, a disease that still claims 400,000 new cases each year. We identified genetic variants in the shared promoter region of the PARK2 and PACRG genes as major risk factors of leprosy susceptibility. Both encoded proteins are part of the cellular ubiquitination system. Specifically, PARK2, the cause of early onset Parkinson's disease, is an E3 ligase that likely is involved in controlled proteolysis, the cellular anti-oxidants response and the regulation of innate immune responsiveness. In addition, numerous E3 ligases have recently been shown to be critical regulators of immunity. While the specific role of PARK2/PACRG in leprosy pathogenesis remains unknown, a number of experimentally testable scenarios can be developed to further explore the role of these proteins in anti-Mycobacterium leprae host responsiveness.     

Primary antitumor immune response mediated by CD4+ T cells

Gene-targeted mice have recently revealed a role for lymphocytes and interferon-gamma (IFNgamma) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNgamma activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages.     

Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis

Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published by December 2004. The limits of detection and efficiency of each real-time PCR assay were determined using DNA extracted from stool samples spiked with serially diluted cultured E. histolytica trophozoites. The ability of each assay to correctly distinguish E. histolytica from E. dispar was evaluated with DNA extracted from patients' stools and liver aspirates submitted for confirmatory diagnosis. Real-time PCR allowed quantitative analysis of the spiked stool samples, but major differences in detection limits and assay performance were observed among the evaluated tests. These results illustrate the usefulness of comparative evaluations of diagnostic assays.     

Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen,but not by the LACK antigen,are protective against experimental Leishmania (Leishmania) amazonensis infection

Leishmania amazonensis is one of the major etiologic agents of a broad spectrum of clinical forms of leishmaniasis and has a wide geographical distribution in the Americas, which overlaps with the areas of transmission of many other Leishmania species. The LACK and A2 antigens are shared by various Leishmania species. A2 was previously shown to induce a potent Th1 immune response and protection against L. donovani infection in BALB/c mice. LACK is effective against L. major infection, but no significant protection against L. donovani infection was observed, in spite of the induction of a potent Th1 immune response. In an attempt to select candidate antigens for an American leishmaniasis vaccine, we investigated the protective effect of these recombinant antigens (rLACK and rA2) and recombinant interleukin-12 (rIL-12) against L. amazonensis infection in BALB/c mice. As expected, immunization with either rA2-rIL-12 or rLACK-rIL-12 induced a robust Th1 response prior to infection. However, only the BALB/c mice immunized with rA2-rIL-12 were protected against infection. Sustained gamma interferon (IFN-gamma) production, high levels of anti-A2 antibodies, and low levels of parasite-specific antibodies were detected in these mice after infection. In contrast, mice immunized with rLACK-rIL-12 displayed decreased levels of IFN-gamma and high levels of both anti-LACK and parasite-specific antibodies. Curiously, the association between rA2 and rLACK antigens in the same vaccine completely inhibited the rA2-specific IFN-gamma and humoral responses and, consequently, the protective effect of the rA2 antigen against L. amazonensis infection. We concluded that A2, but not LACK, fits the requirements for a safe vaccine against American leishmaniasis.     

Dual role of interleukin-4 (IL-4) in pulmonary paracoccidioidomycosis: endogenous IL-4 can induce protection or exacerbation of disease depending on the host genetic pattern

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.     

Immune response in human visceral leishmaniasis: analysis of the correlation between innate immunity cytokine profile and disease outcome

We investigated the cytokine profile of cells of the innate immune response and its association with active (ACT), asymptomatic (AS) and cured (CUR) human visceral leishmaniasis (VL), as well as noninfected (NI) subjects. The frequency of cytokine-producing cells was determined after short-term in vitro incubation of whole peripheral blood samples with soluble Leishmania antigen (SLA). Our data demonstrated a predominant type 2 cytokine profile in NI and ACT. In NI, we observed an increase of IL-4+ neutrophils, IL-10+ eosinophils besides a decrease of tumour necrosis factor (TNF)-alpha+ eosinophils/monocytes. Yet in ACT, we observed an increase of IL-4+ neutrophils and natural killer (NK) cells and IL-10+ monocytes, a reduced frequency of IL-12+ and IFN-gamma+ eosinophils and lower levels of TNF-alpha+ and IL-12+ monocytes. AS presented a mixed profile, characterized by an increase of IFN-gamma+ neutrophils/eosinophils and NK cells, of IL-12+ eosinophils/monocytes, as well as increase of IL-4+ neutrophils and NK cells and IL-10+ eosinophils/monocytes. In contrast, CUR was characterized by a type 1 response with an increase of IFN-gamma+ neutrophils/eosinophils and NK cells, associated with an increase in IL-12+ monocytes. In conclusion, we show a correlation between innate immune cytokine patterns and clinical status of VL, suggesting that these cells, in addition to other factors, may contribute to the cytokine microenvironment in which Leishmania-specific T cells are primed and to disease outcome.