Phenotypic and functional characterization of intestinal epithelial exosomes.

Intestinal epithelial cells (IEC) are located at a strategic position between the external environment and the most extended lymphoid tissue in the body. Besides their central role in the absorption of nutrients, IEC also provide antigenic information to the immune system and are involved in the balance tolerance/allergy to food antigens. Like professional antigen presenting cells, IEC have been shown to secrete 30- to 90-nm diameter vesicles named exosomes, in a polarized way, either from their apical or basolateral side. These vesicles carry molecules involved in adhesion and antigen presentation, comprising major histocompatibility complex (MHC) class I and class II molecules, tetraspan proteins, CD26/dipeptidyl-peptidase IV, and A33 antigen, a molecule essentially restricted to the intestinal epithelium. Invariant chain, transferrin receptor, and Na-K-ATPase are not expressed on epithelial exosomes. In vivo, in mice, epithelial exosomes carrying MHC/ovalbumin peptide complexes induce specific immune responses when injected intraperitoneally. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells and enriched in epithelial exosomes, is found at the surface of cells entering mesenteric lymph nodes suggesting exosome migration from the epithelial layer to the gut associated lymphoid system. Taken together, intestinal epithelial exosomes released at the basolateral surface of enterocytes could be antigen-carrying structures constituting a link between luminal antigens and the local immune system and acting as sensors of the antigenic information present in the intestinal lumen.

Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice.

BACKGROUND: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. METHODS: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon gamma (IFN-gamma) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. RESULTS: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-gamma) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-gamma (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. CONCLUSIONS: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.

Intestinal epithelial cells secrete exosome-like vesicles.

BACKGROUND & AIMS: Given the observations that intestinal epithelial cells (IECs) can present antigens to CD4(+) T lymphocytes and that professional antigen-presenting cells secrete exosomes (antigen-presenting vesicles), we hypothesized that IECs may secrete exosomes carrying molecules implicated in antigen presentation, which may be able to cross the basement membrane and convey immune information to noncontiguous immune cells. METHODS: Human IEC lines HT29-19A and T84-DRB1*0401/CIITA were grown on microporous filters. Release of exosomes under basal or inflammatory conditions was evaluated in conditioned apical and basolateral media after differential ultracentrifugations. Morphologic and biochemical characterization of exosomes was performed using immunoelectron microscopy, Western blotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: The intestinal cell lines released 30-90-nm-diameter vesicles from the apical and basolateral sides, and this release was significantly increased in the presence of interferon gamma. MHC class I, MHC class II, CD63, CD26/dipeptidyl-peptidase IV, and A33 antigen were present in epithelial-derived exosomes. CONCLUSIONS; Human IEC lines secrete exosomes bearing accessory molecules that may be involved in antigen presentation. These data are consistent with a model in which IECs may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.

Evaluation of risk and diagnostic value of quantitative assays for anti-Toxoplasma gondii immunoglobulin A (IgA), IgE, and IgM and analytical study of specific IgG in immunodeficient patients.

To determine their prognostic and diagnostic values for toxoplasmosis in immunodepressed subjects, we assayed immunoglobulin A (IgA) and IgE antibodies by means of immunocapture (IC) tests, with revelation done by using a suspension of T. gondii (ICT). We also carried out a simultaneous analytical study of IgG antibodies on cellulose acetate membranes by using the comparative immunological profile method and an enzyme-linked immunofiltration assay (ELIFA). A total of 1,238 samples (serum, cerebrospinal fluid, and aqueous humor from 318 patients) were tested. IgA and IgE antibodies were detected in all heart, kidney, and liver transplant recipients with clinical manifestations of toxoplasmosis; IgA was detected in the aqueous humor of a patient with chorioretinitis. In patients with AIDS-related toxoplasmosis, including the cerebral form, IgA and IgE antibodies or a significant modification of ELIFA IgG values were observed in 38, 19, and 25% of patients, respectively. IgM was detected by ICT only in 12% of patients and aided the diagnosis in 1 of 71 patients. IC tests for specific IgA and IgE alone and combined with ELIFA were positive in 39 and 46% of patients who developed clinical toxoplasmosis, respectively. In a serial study of 16 patients in whom at least one of these three tests was positive, a significant immunological signal sometimes preceded clinical onset by 1, 6, and even 17 months. Similarly, in a group of human immunodeficiency virus-infected patients with evidence of previous exposure to T. gondii but no clinical manifestations, IgA, IgE, and IgA and/or IgE antibodies were detected in only 11, 4, and 12% of patients, respectively. These two situations point to peripheral T. gondii reactivation. IgA and IgE emerged as interesting markers of the risk of toxoplasmosis in immunodepressed patients. They may also provide valuable assistance in the diagnosis of toxoplasmosis, especially because tests for specific IgM are disappointing. However, at least one in two patients with toxoplasmosis showed no detectable immunological reaction, suggesting that this polyisotypic approach should be combined with other noninvasive methods such as gene amplification.

Anti-P30 IgA antibodies as prenatal markers of congenital toxoplasma infection.

This study extends a previous study and confirms that the detection of anti-P30 IgA antibodies is very helpful in the diagnosis of acute acquired or congenital toxoplasmosis. Moreover, we demonstrate that an anti-P30 IgA response can be mounted in the fetuses infected by Toxoplasma gondii during their intra-uterine life as early as week 23 of gestation. A double-sandwich ELISA described in our previous work was used to detect anti-P30 IgA antibodies in 1378 human serum samples collected from 551 patients, including 162 fetuses whose mothers had been infected by T. gondii during pregnancy, 46 congenitally infected and 90 uninfected newborns and 253 women suspected of having been infected during pregnancy, including the mothers of fetuses and newborns previously described. Anti-P30 IgA antibodies were detected in all cases of acute toxoplasmosis but in no case of chronic toxoplasmosis: in the majority of cases, the IgA antibody titre fell below cut-off in 3-9 months. Among the 46 congenitally infected newborns, anti-P30 IgA antibodies were detected in sera of 41 infected newborns (38 at birth, two in the first months of life, one in the seventh month of life), while anti-P30 IgM antibodies were detected in only 30 cases at birth and in one case during the first month of life. Among 162 fetuses, anti-P30 IgA response was observed in five infected fetuses, but was not detected in either 152 uninfected fetuses or in five fetuses considered as infected. The absence or presence of anti-P30 IgA antibodies in the fetus is discussed in relation to the date of maternal infection and collection of the fetal blood. It clearly appears from our study that the combined testing of both IgM and IgA in the fetus and the newborn is essential for a more efficient diagnosis of infection.

What is known about the prevention of congenital toxoplasmosis?

The French programme for the prevention of congenital toxoplasmosis consists of the diagnosis and treatment with spiramycin of acute infections during pregnancy and monthly follow-up of all identified seronegative women. The major flaw is that the efficacy of spiramycin in preventing contamination of the fetus, or at least in reducing the extent of the infection, has never been evaluated in a randomised placebo-controlled clinical trial. Its evaluation would require the follow-up of children born to mothers contaminated during pregnancy for more than 6 months, a goal that is difficult to obtain in current practice. The cost of the programme depends largely on the proportion of non-immune women of childbearing age. Since the modes of contamination are known and are linked to living habits, it should be possible to reduce the risk of infection during pregnancy by adequate health education. This approach is still to be evaluated.

Comparative biological activities of the four synthetic (5,6)-dihete isomers.

(5,6)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids [5,6)-DiHETEs) were synthesized and separated into four pure diastereoisomers. They were tested for comparative binding affinities to leukotriene receptors (LTC4, LTD4, LTB4) in guinea pig lung membranes. Only (5S,6R)-DiHETE was recognized by the LTD4 receptor, the other receptors interacted with neither of the four isomers. (5S,6R)-DiHETE also contracted ileum in vitro and this effect was inhibited by the LTD4 receptor antagonists ICI 198,615 and SKF104,353. These data suggest that the bioproduct (5S,6R)-DiHETE generated by enzymatic conversion of LTA4 could have some LTD4-like activity when produced in large concentrations.

Synthesis and contractile activity of new acetylenic and allenic analogues of leukotrienes C4 and D4: importance of the Z-11,12 double bond.

The (5S,6R) isomers of new acetylenic and allenic analogues of leukotrienes C4 and D4 were synthesized for comparative pharmacological studies on intestinal smooth muscle preparations. These new analogues are poor spasmogenic agonists, the replacement of the 11,12-ene with a relatively more stable triple bond causing an important reduction in intrinsic activity. They did not show any significant antagonist activity. Unexpectedly, these results prove that the 11,12 portion in the triene structure of the lipophilic chain is critical for an agonist activity.

Epidemiology of toxoplasmosis among pregnant women in the Paris area.

A survey was performed using a sample of pregnant women selected at one of the biggest test centres in the Paris area. These women were serologically screened for toxoplasmosis between October 1981 and September 1983 (according to the prevention protocol for congenital toxoplasmosis established by the French health ministry). The prevalence rate of specific antibodies for toxoplasmosis was estimated from the 1074 women who were tested for the first time during the study. The prevalence rate among pregnant women in the Paris area was derived by direct standardization according to age and geographical origin. A standardized prevalence rate of 71% +/- 4% among French women, of 51.4% +/- 5% among immigrant women and a global adjusted prevalence rate of 67.3% +/- 3% for pregnant women in the Paris area was found. An incidence rate of 1.6% was estimated for the 2216 non-immune pregnant women included in the sample. There is no significant difference between the probabilities of seroconversion among French and immigrant women (2.3% +/- 1% and 1.6% +/- 0.8% respectively). Comparison of the data with previous study results show a decrease in the prevalence rate of specific antibodies for toxoplasmosis in the Paris area over the last 20 years that cannot be explained by changes in age and geographical origin. No data were available to support an aetiological hypothesis for a decrease in toxoplasma transmission to humans. Since immigration and a decrease in toxoplasma transmission to humans has led to a larger population of women at risk of infection during pregnancy in France, it is therefore important to perform studies to investigate risk factors and markers of acquired toxoplasmosis during pregnancy in order to improve the prevention of congenital toxoplasmosis.

[Serodiagnosis of toxoplasmosis developing during pregnancy and of congenital toxoplasmosis]. / Sérodiagnostic de la toxoplasmose évolutive en cours de grossesse et de la toxoplasmose congénitale.

During the first prenatal serodiagnosis of toxoplasmosis, the test must permit to differentiate between immunized and non-immunized patients and to screen recently contracted toxoplasmosis. In a group of 33 women affected with toxoplasmosis during pregnancy a critical study of serodiagnosis criteria is carried out by comparing the theoretical protocol of the evolution of the serology during acquired toxoplasmosis with the situations observed under usual prenatal monitoring. Seroconversion was noted in 26 women and the variability of the results emphasizes the difficulties in determining the date of the contamination when an evolutive form of toxoplasmosis is suspected at the first examination, which is the case in 7 other patients. In children, 11 congenital toxoplasmosis were diagnosed, all on laboratory examinations. It must be emphasized that 16 children were prematurely lost to follow-up. It should be necessary to devote our energies to screening and information in order to validate the protocol of prevention of congenital toxoplasmosis.

[Transplacental passage of toxoplasma antibodies. The difference between African and European mothers]. / Passage transplacentaire des anticorps toxoplasmiques. Différence entre la mère africaine et européenne.

The transplacental transfer of toxoplasmosis antibodies has been compared between european and african cord/mother ratios. A lack of transfer is statistically noticed among african cord-mother pairs. This transfer deficiency is highly significantly negative correlated with mother total IgG level.

[Hepatic amebiasis at Niamey Hospital (Nigeria)]. / L'amibiase hépatique à l'hôpital de Niamey (Niger).

The authors report the results of a prospective study on hepatic amebiasis realized in national hospital of Niamey (Republic of Niger). Hepatic amebiasis is uncommon in Niger as in other hot and dry countries of Africa whereas it is common in hot and damp areas of the continent. These results point out the hypothesis of a hepatotropic strain of Entamoeba histolytica uncommon in Sahelia area whereas the amebiasis infestation is frequent.

Effects of pefloxacin on phagocytosis function of rat macrophages and polymorphonuclear leucocytes.

The influence of pefloxacin on polymorphonuclear leucocytes (PMN) and alveolar macrophages (AM) phagocytosis and microbicidal activity was studied. Phagocytes were collected from Sprague-Dawley rats, mixed with pefloxacin at different concentrations (1, 10, 100 mg/l) and a yeast phagocytosis assay was performed. Three parameters were measured, the phagocytic capacity (number of live and dead yeast/100 cells), the phagocytic activity (number of cells containing 2 or more yeast/100 cells) and the microbicidal activity or % killed (Formula: see text) Both low and high concentrations of pefloxacin increased significantly the phagocytic capacity and activity of AM and PMN. The increase of the AM phagocytic capacity and activity seen with increasing levels of extracellular pefloxacin was not statistically significant. Pefloxacin showed no adverse effect on the microbicidal activity of AM and PMN.

Specificity of acebutolol-induced antinuclear antibodies.

Treatment with the beta blocker acebutolol may trigger antinuclear antibody (ANA) production. We retrospectively studied 97 sera from 47 patients who developed ANA during acebutolol treatment. Anti-histone and anti-denatured (ss) DNA antibodies were found in 53% and 66% respectively of the sera tested. The activities of these two antibodies correlated well with the total ANA titer. Anti-native (ds) DNA were absent or present at low titer. This immunochemical pattern of acebutolol-induced ANA is similar to that reported for other drug-induced ANA. To date, the presence such isolated ANA is not known to expose patients to any particular risk other than exceptional and minor clinical manifestations of lupus which are rapidly reversible following therapy cessation.

[Hepatic amebiasis: various aspects in Brazzaville]. / L'amibiase hépatique: les différents aspects à Brazzaville.

The authors present the results of a study of 27 cases of hepatic amoebiasis observed in the general hospital of Brazzaville from the 1st January to the 30th June 1982; then they precise the various aspects of this frequent infection in Congo.

[Incidence of antinuclear antibodies during treatment with acebutolol. A 1-year prospective study in 45 hypertensive patients treated by monotherapy]. / Incidence des anticorps antinucléaires au cours des traitements par acébutolol. Etude prospective sur 1 an chez 45 hypertendus traités en monothérapie.

Determinations of antinuclear antibodies (ANA) were performed before treatment, then every 3 months for one year, in 45 hypertensive patients treated with acebutolol alone in daily doses equal or superior to 800 mg. Non significant ANA titers (1/10 to 1/40) were detected before treatment in 8.8% of the patients. After one year of treatment, 15.5% of the patients were found to have antinuclear antibodies. In 4 (8.8%) female patients (3 of whom were ANA-negative before treatment) the ANA titers were above 1/40. Treatment was discontinued in one of these due to the occurrence of bronchospasm. No lupus-like syndrome was observed during the course of the study. Prolonged prospective studies with acebutolol alone would be required to determine whether in the long term the incidence of significant ANA titers remains at the 8.8% level or increases.