Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells.     

In vitro and in vivo studies of macrophage functions in amebiasis.

Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets.     

Activation of macrophages by Entamoeba histolytica extracts in mice

The effect of Entamoeba histolytica extracts on the production of inflammatory macrophages and the release of hydrogen peroxide (H2O2) and superoxide (O2-) from these cells was examined in C57BL/6 mice. Two different strains of E. histolytica, either virulent (IP:0682:1) or nonvirulent (DKB), were used in this study. The number of macrophages recovered from the peritoneal cavity of mice treated 5 days previously with 150 micrograms of either strain of amoebic extracts was significantly higher than in the saline-treated groups. Macrophages from mice treated with 150 micrograms of the IP:0682:1 strain of amoebic extracts exhibited a powerful burst of oxidative metabolis when triggered with phorbol myristate acetate (PMA). Extract from the non-virulent strain was much less effective in activating macrophages for the production of oxidative metabolites. Thus, a crude extract preparation from E. histolytica, particularly the virulent strain, induces a strong macrophage inflammatory response in which the cells also produce high levels of H2O2 and O2-.     

The tumor necrosis factor alpha-stimulating region of galactose-inhibitable lectin of Entamoeba histolytica activates gamma interferon-primed macrophages for amebicidal activity mediated by nitric oxide.

Entamoeba histolytica adheres via galactose-lectin (Gal-lectin) to human colonic mucins and intestinal epithelial cells as a prerequisite to amebic invasion. Native Gal-lectin is a protective antigen in the gerbil model of amebiasis. Amino acids 596 to 1082 of Gal-lectin mediate E. histolytica adherence to target cells and stimulate tumor necrosis factor alpha (TNF-alpha) production by naive murine bone marrow macrophages (BMM). Resistance to amebiasis requires an effective cell-mediated immune response against E. histolytica trophozoites mediated by nitric oxide (NO) released from activated macrophages. Herein, we determine whether the TNF-alpha-stimulating region of Gal-lectin can activate gamma interferon (IFN-gamma)-primed BMM for NO production and amebicidal activity. Native Gal-lectin (100 to 500 ng/ml) stimulated TNF-alpha and inducible nitric oxide synthase (iNOS) mRNA expression in IFN-gamma-primed BMM as did lipopolysaccharide (100 ng/ml). Primed BMM produced TNF-alpha and NO in response to Gal-lectin in a dose-dependent manner. Antilectin monoclonal antibody IG7, which recognizes a domain (amino acids 596 to 818) of the TNF-alpha mRNA-stimulating region of Gal-lectin, specifically inhibited TNF-alpha and iNOS mRNA induction and TNF-alpha and NO production by primed BMM in response to Gal-lectin (100 ng/ml). Simultaneous treatment of BMM with IFN-gamma and Gal-lectin (100 ng/ml) activated the cells to kill E. histolytica trophozoites, whereas IFN-gamma treatment alone had no effect. In the presence of monoclonal antibody 1G7 or aminoguanidine (an iNOS inhibitor), NO production and amebicidal activity were inhibited >80%. These results suggest that the TNF-alpha-stimulating region of native Gal-lectin is a potent stimulus of IFN-gamma-primed BMM for NO production, which is essential for host defense against amebiasis.     

Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

AIMS--To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS--Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS--Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS--Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.     

Antibody-dependent macrophage-mediated cytotoxicity against Entamoeba histolytica

Interactions between trophozoites of Entamoeba histolytica and peritoneal exudate macrophages from unsensitised and antigen-sensitised animals were studied in vitro. Normal macrophages killed trophozoites to some extent. This killing capacity was enhanced by prior sensitisation of the animals with specific antigen. Incorporation of anti-amoebic antiserum in the amoeba-macrophage mixture greatly enhanced the killing capacity of macrophages. Fraction one (F-I) of a crude amoebic extract was most effective in enhancing the cytotoxicity of macrophages by prior sensitisation and anti-F-I serum was the most effective antiserum. The cytotoxicity-inducing capacity of the immune serum resided in the IgG but not in the IgM fraction.     

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.     

Roles of cell adhesion and cytoskeleton activity in Entamoeba histolytica pathogenesis: a delicate balance

The protozoan parasite Entamoeba histolytica colonizes the human large bowel. Invasion of the intestinal epithelium causes amoebic colitis and opens the route for amoebic liver abscesses. The parasite relies on its dynamic actomyosin cytoskeleton and on surface adhesion molecules for dissemination in the human tissues. Here we show that the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin clusters in focal structures localized in the region of E. histolytica that contacts monolayers of enterocytes. Disruption of myosin II activity impairs the formation of these structures and renders the trophozoites avirulent for liver abscess development. Production of the cytoplasmic domain of the E. histolytica Gal/GalNAc lectin in engineered trophozoites causes reduced adhesion to enterocytes. Intraportal delivery of these parasites to the liver leads to the formation of a large number of small abscesses with disorganized morphology that are localized in the vicinity of blood vessels. The data support a model for invasion in which parasite motility is essential for establishment of infectious foci, while the adhesion to host cells modulates the distribution of trophozoites in the liver and their capacity to migrate in the hepatic tissue.     

In vitro effect of human recombinant tumor necrosis factor on Entamoeba histolytica trophozoites

The effect of tumor necrosis factor (TNF) on E. histolytica trophozoites was examined by using three virulent (IP: 0682:1, HM-1: IMSS, 200: NIH) and one nonvirulent (DKB) strain of E. histolytica. Various concentrations of recombinant TNF were added to E. histolytica trophozoites and the total parasite numbers and their viability were periodically assessed by microscopic observation and trypan blue staining after incubation at 37 degrees C in a nonhumidified chamber. In this study, concentrations of 10(1)-10(6) units of TNF were used. Over a concentration range of 10(4)-10(6) units, the number of trophozoites was significantly lowered in the amoebic cultures containing TNF as compared to untreated controls. It was also found that the effect of TNF was dependent on the densities of both virulent and non-virulent strains of E. histolytica trophozoites in axenic conditions. TNF has no significant affect on the growth of amoebae at the lower starting number of amoebae. The amoebae cultured at the higher density were growth-inhibited significantly in comparison with the control groups. When the growth of the virulent and nonvirulent strains of amoebae was compared in TNF treated culture, it was found that TNF has an inhibitory effect on both the virulent and nonvirulent strans of E. histolytica.     

Cytopathic changes in rat microglial cells induced by pathogenic Acanthamoeba culbertsoni: morphology and cytokine release.

To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-alpha secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1beta secreted from microglial cells cocultured with A. culbertsoni trophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-alpha and IL-1beta from microglial cells.     

Detection and identification of entamoeba species in stool samples by a reverse line hybridization assay

Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.     

Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo.

Entamoeba histolytica infections are associated with a state of transient suppression of cell-mediated immunity. Macrophages, the most important cells in host defence and control of invasive amoebiasis, in infected animals have been found to be deficient in effector functions and accessory cell potential. However, little is known of the cellular mechanisms responsible for the down-regulation. This study investigated whether macrophage dysfunction in amoebiasis is associated with altered macrophage arachidonic acid (AA) metabolism. Resident peritoneal macrophages (PMO) from naive gerbils produced enhanced levels of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in response to live E. histolytica trophozoites, to diffusible excretory/secretory products released from live amoebae in millicells and to freeze-thawed soluble amoebic proteins that were inhibitable by indomethacin (INDO) and nordihydroguaiaretic acid (NDGA), respectively. In contrast to PMO from naive gerbils, PMO from animals with amoebic liver abscesses at 10, 20 and 30 days post-infection (p.i.) released high basal levels of PGE2 and LTC4. In response to zymosan stimulation, PMO from infected animals produced two- and fourfold less PGE2 and LTC4, respectively, as compared to uninfected controls. AMO showed high constitutive basal release of PGE2 and LTC4. In response to amoebic and zymosan stimulation, AMO at 10 days p.i. produced significantly higher levels of PGE2 than AMO at 20 days p.i., while AMO at 30 days p.i. were refractory to stimulation to produce higher than basal levels of PGE2. Early (10 days) and late (20-30 days) AMO were refractory to amoebic and zymosan stimulation for enhanced LTC4 release. Pretreatment of AMO with AA substrate restored optimal PGE2 and LTC4 biosynthesis, but the cells were generally unresponsive to zymosan stimulation to produce augmented levels of LTC4. These results strongly suggest that intrinsic or secreted products or both from E. histolytica can induce profound alteration of eicosanoid formation in cyclooxygenase and lipoxygenase pathways in macrophages from naive and infected animals and that AA metabolism by AMO is sequentially modified during the course of the disease.     

Characterization of anti-islet cytotoxic human T-cell clones from patients with type 1 diabetes mellitus

To identify important anti-islet T-cells and their target antigen(s), we have isolated and characterized seventeen human T-cell clones which are reactive to an extract of rat insulinoma (RIN) cells from three children with new onset type 1 diabetes mellitus (T1D). Of these 17 clones, 15 were found tissue specific. Six of eight tested tissue specific clones did not recognize known islet antigens such as GAD, 52 kDa islet protein, insulin, ICA512, and heat shock protein 60 (hsp60), suggesting that these clones recognize an autoantigen not previously identified. All tested clones were phenotypically CD4 and functionally Th0 or Th0/Th1 cells. One RIN extract reactive clone (2E9) recognized hsp60 and was CD4 and TCR α/β positive. This clone also proliferated in response to human and rat islets suggesting that the antigen is conserved between species. This clone and 75% of all the tested RIN reactive clones exhibited anti-islet cytotoxicity by lysing target cells coated with RIN extract. HLA DR determinants may play a role in this cytotoxic activity since preincubation with HLA DR antibody decreased the anti-islet cytoxicity of the two tested clones. In conclusion, we have isolated RIN reactive CD4+T-cell clones from diabetic subjects, six of which appears tissue specific and non-reactive to putative important islet antigens, and in turn may be recognizing yet undiscovered islet antigens. The high frequency anti-islet cytotoxic properties of the islet reactive clones provides evidence for a role of CD4+ cytotoxic T-lymphocytes in the diabetic process. Further, the isolation of hsp60 reactive clone with anti-islet cytotoxic properties suggests that cell mediated immunity against hsp60 may be important in the pathogenesis of diabetes.     

Human neutrophils activated by interferon-gamma and tumour necrosis factor-alpha kill Entamoeba histolytica trophozoites in vitro

The interaction between cytokine-activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rIFN-gamma alone allowed them to kill 30% of E. histolytica trophozoites; however, rIFN-gamma and rTNF-alpha pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rIFN-gamma-treated neutrophils were shown to kill 70% of amebae, and rIFN-gamma- and rTNF-alpha-treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with increased neutrophil resistance to amebic contact-dependent killing and was shown to be 73% H2O2 dependent.     

Characterization of anti-islet cytotoxic human T-cell clones from patients with type 1 diabetes mellitus

To identify important anti-islet T-cells and their target antigen(s), we have isolated and characterized seventeen human T-cell clones which are reactive to an extract of rat insulinoma (RIN) cells from three children with new onset type 1 diabetes mellitus (T1D). Of these 17 clones, 15 were found tissue specific. Six of eight tested tissue specific clones did not recognize known islet antigens such as GAD, 52 kDa islet protein, insulin, ICA512, and heat shock protein 60 (hsp60), suggesting that these clones recognize an autoantigen not previously identified. All tested clones were phenotypically CD4 and functionally Th0 or Th0/Th1 cells. One RIN extract reactive clone (2E9) recognized hsp60 and was CD4 and TCR alpha/beta positive. This clone also proliferated in response to human and rat islets suggesting that the antigen is conserved between species. This clone and 75% of all the tested RIN reactive clones exhibited anti-islet cytotoxicity by lysing target cells coated with RIN extract. HLA DR determinants may play a role in this cytotoxic activity since preincubation with HLA DR antibody decreased the anti-islet cytoxicity of the two tested clones. In conclusion, we have isolated RIN reactive CD4+T-cell clones from diabetic subjects, six of which appears tissue specific and non-reactive to putative important islet antigens, and in turn may be recognizing yet undiscovered islet antigens. The high frequency anti-islet cytotoxic properties of the islet reactive clones provides evidence for a role of CD4+ cytotoxic T-lymphocytes in the diabetic process. Further, the isolation of hsp60 reactive clone with anti-islet cytotoxic properties suggests that cell mediated immunity against hsp60 may be important in the pathogenesis of diabetes.     

Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica.     

Immunoprotective behaviour of plasma-membrane-associated antigens of axenic Entamoeba histolytica

Immunisation of golden hamsters with plasma-membrane-associated antigens of a virulent subline of axenic Entamoeba histolytica strain NIH 200 V, entrapped in multilamellar phosphatidylcholine liposomes (MPL) or Freund's complete adjuvant (FCA), afforded protection against intrahepatic challenge with axenic amoebic trophozoites of the same strain. Amoebic liver abscess developed in 86% and 80% of the animals that received empty liposomes or buffer emulsified in FCA but in none of the animals that received plasma-membrane-antigen vaccines. All the immunised animals had significantly higher levels (p less than 0.001) of antibodies to plasma-membrane components and significantly higher levels (p less than 0.001) of cellular sensitisation. Antibody-dependent macrophage-mediated cytotoxicity against trophozoites was also found to be significantly greater (p less than 0.001) in immunised animals. Liposome-entrapped antigens stimulated the immune system of the host as well as, or better than, antigens administered with FCA.     

In vivo and in vitro characterization of murine T-cell clones reactive to Mycobacterium tuberculosis.

Seventeen helper T-cell clones were derived by stimulating lymph node cells from sensitized C57BL/6 mice with Mycobacterium tuberculosis H37Ra, M. tuberculosis H37Rv, or purified protein derivative. Most clones cross-reacted with Mycobacterium bovis BCG, H37Ra, H37Rv, and purified protein derivative. However, four clones were able to differentiate H37Rv from H37Ra, or BCG from H37Ra and H37Rv. In addition, four other T-cell clones recognized recombinant antigens of 19 and 65 kilodaltons isolated from a genomic expression library of M. tuberculosis by using monoclonal antibodies. All clones were Ia restricted and had the Thy-1.2+ Lyt-1+ L3T4+ Lyt-2- phenotype. On stimulation with antigen, all of the clones tested secreted interleukin-2 and gamma interferon but not B-cell stimulatory factor 1. All of the clones tested induced an antigen-specific delayed-type hypersensitivity response upon local cell transfer, although the magnitude of this response differed markedly among clones.     

Development of a diagnostic test for Entamoeba histolytica using idiotype expression in human

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis.Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites.Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.     

Function and antigen recognition pattern of L3T4+ T-cell clones from Mycobacterium tuberculosis-immune mice.

T-cell clones were established from Mycobacterium tuberculosis-immunized mice. These clones had the phenotype Thy-1+ L3T4+ Lyt-2- and were restricted by the H-2I-A locus. After antigen stimulation, the T-cell clones secreted interleukin-2 and gamma interferon. Factors produced by these T-cell clones activated normal bone marrow macrophages for antimycobacterial activity in vitro. Furthermore, the T-cell clones could adoptively confer delayed-type hypersensitivity on normal recipient mice. These findings indicate that the T-cell clones clones expressed relevant functions of antimycobacterial immunity. The antigen reactivity of the T-cell clones to different mycobacterial species ranged from broad cross-reactivity to stringent specificity, and none of the clones distinguished between M. tuberculosis and M. bovis. Thus, M. tuberculosis-immune helper/inducer T cells of identical phenotype, genetic restriction, and function varied in their antigen specificity. T-cell clones of the type described will facilitate functional characterization of mycobacterial antigens on the T-cell level.