Periodicity and patterns of Entamoeba histolytica and E. dispar infection in HIV+/AIDS patients in Mexico.

In a 12-month longitudinal study, a cohort of Mexican HIV+/AIDS patients was checked several times for Entamoeba infection, with the parasites identified, as E. histolytica or E. dispar, using PCR. The polymorphic region of the parasites' chitinase genes was investigated by PCR, with the variation in amplicon sizes being used as a measure of the genetic variation among the isolates. The patients found infected with Entamoeba at the start of the study displayed varied patterns of infection clearance and re-infection. The analysis of the polymorphisms in the chitinase gene revealed seven polymorphic patterns in the E. histolytica isolates investigated and three in the E. dispar isolates. Many of the patients were each re-infected with Entamoeba at least once during the 12 months of follow-up. As seen in a previous study in Mexico, none of the E. histolytica-infected patients developed any clinical symptoms of invasive amoebiasis during the follow-up period. The results highlight the complexity of the host-parasite relationship in human amoebiasis.

Persistence of secretory antiamoebic antibodies in patients with past invasive intestinal or hepatic amoebiasis.

In the present work, it was demonstrated that in amoebic dysentery and amoebic liver abscess patients, the secretory response is long-lasting (> 12 months); and 50% of amoebic dysentery patients developed circulating antiamoebic IgG in comparison with 100% of amoebic liver abscess individuals. A total of 83% of these individuals developed high levels of serum anti-Entamoeba histolytica IgA. However, only 10.4% of the dysentery patients showed this anti-E. histolytica antibody isotype in serum. There was no correlation between secretory and serum antiamoebic response, suggesting independent inductive and effector sites in both compartments.

The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica.

Formalin fixation is the most common storage, transportation and preservation method for stool samples. However, fixation dramatically reduces our ability to extract from stool samples DNA that is a suitable template for polymerase chain reaction (PCR)-based diagnostic tests. In this study we evaluated the effects of formalin concentration and of the time stored in fixative on the success of PCR amplification. We found that the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification.

HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica/E. dispar.

The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLA-B, and HLA-DR profiles of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identified. Entamoeba species were identified through zymodeme patterns and/or amplification of species-specific DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no specific HLA marker is associated with the asymptomatic cyst passers' condition. These findings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no significant association with amebic rectocolitis was found.

Prospective study of Entamoeba dispar infection in a cohort of mothers and their infants: relationship to serum antibody response.

The sera of cohorts of newborn infants and their mothers, characterized as cyst passers of Entamoeba with nonpathogenic zymodemes (E. dispar) and seropositive for amoebic antigens, were analyzed. Both cohorts were followed for a period of 12 months by microscopic examination of feces and determination of serum anti-amoebic antibody titers using the indirect hemagglutination assay. Control groups (noncyst passer mothers and their infants) were included and followed. To characterize antigens involved in the induction of IgG and IgA antibody responses, Western blots of serum from all participants were tested and immunoplots of the frequency of antigenic recognition were constructed. Results of clinical follow-up and microscopic examination of feces showed that during the 12-month period none of the cyst passer mothers had episodes of diarrhea attributable to E. histolytica invasion; five of 21 children of cyst passer mothers became infected during the study, five of five infected children developed serum antiamebic antibodies (titers 1:64-1:128); none of the cohort of children from cyst passer mothers had diarrhea due to E. histolytica. Western blot analysis showed that there are antigenic fractions that induce serum antibodies of the IgG and IgA classes against E. dispar very early in the host-parasite relationship. Our results suggest that mechanisms of antibody induction different from intestinal invasion may be operating in amebic infection. Intestinal absorption of antigen, systemic reflection of secretory antibody response, and priming of newborns by maternal anti-idiotypic antibody transfer are discussed.

Human cell-mediated immune responses to antigenic fractions of Salmonella typhi.

The proliferative responses of peripheral blood mononuclear cells of 10 subjects that had typhoid fever, and healthy volunteers without history of typhoid fever or immunization against disease, were analysed with antigen fractions from two protein extracts of Salmonella typhi. Fractions from each extract were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred to nitrocellulose filters by electroblotting and processed to obtain antigen-bearing nitrocellulose particles for use in lymphocyte cultures. Although the individual proliferative responses were heterogeneous we identified two main immunogenic regions of 29-32 10(3) MW and 45-56 x 10(3) MW for both extracts. Even though there was no one particular antigenic fraction capable of stimulating lymphocytes from all individuals with a previous history of typhoid fever, the combination of three fractions 29-32, 41-45, 63-71 x 10(3) MW could be stimulatory for cells of 90% of these individuals. Also, four subjects that did not respond to unfractionated antigens gave proliferative responses to several fractions of the same extract. We have identified the main immunogenic fractions of S. typhi that might play a role during typhoid infection and postinfection immunity, and merit further purification and characterization.

Cellular immune response to fractionated avian antigens by peripheral blood mononuclear cells from patients with pigeon breeder's disease.

Pigeon breeder's disease (PBD), a form of hypersensitivity pneumonitis caused by repeated inhalation of antigens of pigeon origin, is characterized by a diffuse inflammation of the lower respiratory tract. Although a variety of immunologic and nonimmunologic mechanisms have been described in the development of the disease, the pathogenesis is still far from clear. In this study we analyzed the T-lymphocyte proliferative response to a variety of avian antigens with use of peripheral blood mononuclear cells from 11 patients who had PBD and 10 healthy volunteers. We used a new method based on avian antigen-bearing nitrocellulose particles derived from Western blots to study the T-cell proliferative response to 15 antigenic fractions obtained from pigeon serum. With this technique, complex mixtures of antigens can be fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and used for T-cell proliferation assays with selected antigenic determinants. A wide variety of responses were observed, and there were no reproducible patterns of reaction within either group. Nine of 10 healthy subjects responded to some soluble fractions. However, patients with PBD displayed the strongest response and responded to a significantly greater number of antigenic fractions. Fraction 2, representing a 220 kd molecular weight protein, was the only immunodominant antigen when both groups were compared; it was recognized by 73% of the patients with PBD and by only 20% of control subjects (p < 0.03). These findings show that T lymphocytes of patients with PBD recognize a wide range of bird proteins, which induce marked T-cell proliferation.

Salmonella typhimurium infection in mice: lymphoproliferative responses to fractionated protein antigens.

The proliferative response of spleen cells of mice immunized with S. typhimurium by the oral route was analyzed using antigen fractions from a protein extract of the bacteria. Mice that survived the challenge with a virulent strain of Salmonella and normal mice were also studied. Mice were immunized with three doses of live S. typhimurium on consecutive days (3C) or once a week for 3 weeks (3W). Fractions 12-100 kDa from the protein extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and processed to obtain particulate antigens for use in lymphocyte cultures. Mice immunized weekly showed a higher survival rate and responded to more antigenic fractions. We identified three fractions of 68-76, 50-52, and 42-45 kDa that were immunodominant for spleen cells from S. typhimurium immunized mice and from survivors to the challenge with the virulent strain.

Entamoeba histolytica: antibody response to recent and past invasive events.

Sero-epidemiological data from endemic amoebiasis areas are difficult to evaluate because the serology of individuals affected by an active process of Entamoeba histolytica tissue invasion is, at present, almost impossible to distinguish from that of individuals who have had an invasive event in the past. The present study compares serum antigenic recognition frequencies among three groups of individuals with different infective conditions: amoebic liver abscess patients; asymptomatic cyst passers; and individuals who have had amoebic liver abscess from one to three years before the study. Control groups consisted of Mexican and Canadian healthy adults. Western blots of E. histolytica membrane extract antigen were reacted with sera from the studied individuals, recognition frequency values were calculated and immunoplots of frequency differences were constructed. The results obtained suggest that the identification and purification of antigenic fractions, which are frequently recognized by sera of amoebic liver abscess patients (136, 132, 93, 70 and 62 kDa), or preferentially associated with past invasive events (144, 140 and 49 kDa), or related to the E. histolytica cyst passer condition (62 and 136 kDa), are important improvements in the use of serology for diagnosis and epidemiological studies in endemic areas of amoebiasis.

Western blot of Entamoeba histolytica antigenic fractions: reactivity analysis with sera from intestinal amoebiasis patients.

The reactivity of sera from acute-phase intestinal amoebiasis patients (two weeks evolution) was studied to determine which of the Entamoeba histolytica antigens are most frequently immunogenic. Sera were examined by means of immunoelectrotransferase assay using crude extract of HM1:IMSS E. histolytica trophozoites. Three populations of clinically healthy adults from Mexico, Canada and Germany, with no evidence of parasites in faeces, were used as controls. The frequency of antigen recognition was analysed. In ailing individuals, the bands of 23, 24, 26 and 51 kDa were recognized most frequently (65 and 60%) followed by the 62 kDa band (56%). The combination of some of these bands, namely 3.4, 4.1 and 6.7, with molecular weights of 62, 51 and 24 kDa, increased the recognition frequency of patients to 91.4%. These results constitute a first but important step towards the design of more accurate methods for the successful immunodiagnosis and epidemiology of acute intestinal amoebiasis.

Local and systemic antibody response in Balb/c mice immunized with Entamoeba histolytica trophozoites.

Several immunization schedules with E. histolytica trophozoites were tested on Balb/c mice in order to induce antibody responses, both in intestinal secretions and in serum. Mice were immunized either orally, systemically, or using one of two combined schedules: the oral route followed by the systemic route (footpad), or vice versa. Each of the immunization schedules used in this project induced an anti-E. histolytica antibody response and there appears to be a correlation between the immunization route employed and the immunoglobulin isotype induced in the gut. Secretory IgA production is favored by the oral administration of trophozoites, whereas mucosal IgG appears to be enhanced by the systemic immunization route. Both schedules are effective in the induction of secretory IgA in the gut, yet higher and earlier levels of IgA appear in orally immunized mice. When systemic immunization is employed, the increase in antibody levels in the intestinal fluid is slower, and IgG is the predominant class. The combined oral/systemic routes of immunization appear to be comparably effective for the induction of local and systemic IgA and IgM antibody production. However, mice immunized first systemically and then locally produce more IgG in both compartments. Combined schedules modify the isotype pattern of antibody responses in serum and in intestinal secretions when compared with single (i.e., oral or systemic) schedules, but they do not appear to favor a secretory IgA immune response.

Inhibition of adhesion process mediated by anti-Entamoeba histolytica specific monoclonal IgA antibodies.

Adhesion of trophozoites to target cells in the host intestine is the first of three consecutive steps (adherence, cytolytic effect and phagocytosis) involved in the invasion of colonic tissue by E. histolytica. To investigate the possible participation of the local secretory immune response in the interference with this early host-parasite relationship, we produced IgA monoclonal anti-E. histolytica antibodies to test their capacity for blocking the adhesion process in vitro (MDCK and HT-29 cell lines) and in situ (colonic mucosa from Balb/c mice and gerbils). Results demonstrate that the monoclonal antibodies tested inhibited trophozoite adherence both in vitro and in situ. This suggests that the local antibody response may play an important role in protection against the invasive process in intestinal amebiasis.