Hemoprotozoa of domestic animals in France: prevalence and molecular characterization.

Very limited information is available on epizootiology of haematozoan infections in French domestic animals. In an attempt to address this issue, prevalence of piroplasmida was studied in carnivores and ruminants, whereas prevalence of Hepatozoon spp. was only investigated in carnivores. In total, 383 animals were included in the survey (namely 116 cats, 108 dogs, 91 sheep and 68 cows). Parasite diagnosis was carried out using molecular methods such as PCR and sequencing of the 18S rRNA gene. In addition, ruminant samples were analyzed with the reverse line blotting technique (RLB). Results of RLB and PCR plus sequencing were in total agreement. In carnivores, haematozoan prevalence was close to 1%. Two cats were infected by H. canis (1.7% prevalence) and one of them was co-infected by Cytauxzoon sp. (0.8%). This represents the first finding of both pathogens in French cats. One dog was infected by H. canis (0.9%) and another by Babesia canis vogeli (0.9%). In ruminants, haematozoan prevalence (piroplasmida) was significantly higher than in carnivores (4.8% in sheep and 8.8% in cow). Theileria ovis was found in 1 sheep, Theileria sp. in 2 sheep, Theileria buffeli in 5 cows and B. major in 1 cow. Evidence presented in this contribution indicates that haematic protozoa are not widely distributed in domestic mammal populations of France.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

IFN-gamma induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicity.

It has been reported that interferon (IFN)-gamma should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-gamma, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis, Moreover, IFN-gamma treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-gamma-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-gamma may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-gamma embryotoxicity.

Fas-mediated apoptosis of mouse embryo stem cells: its role during embryonic development.

PROBLEM: Fas antigen (APO-1/CD95) can regulate the activity of various cells during adulthood. This study aimed at determining whether Fas may also be involved in the regulation of very early events such as the embryo preimplantation stage. METHOD OF STUDY: We used mouse embryo stem (ES) cell line as a model for testing the effect of Fas crosslinking upon anti-Fas monoclonal antibody (MoAb) treatment. In addition, this treatment was also applied to in-vitro mouse-embryo culture. RESULTS: Flow-cytometry analysis of cultured ES cells demonstrated an increase in Fas expression. unchanged in the presence of mouse interleukin-2, while greatly upregulated in the presence of lipopolysaccharide (LPS). As determined by various means, ES cells may undergo a Fas-mediated apoptosis, slightly but significantly intensified by the addition of LPS to cell cultures. We also report that anti-Fas MoAb directly inhibited two-cell stage mouse-embryo (preimplantation) development in in-vitro culture conditions. CONCLUSION: These data suggest a novel mechanism controlling the regulation of physiological cell turnover as well as blastocyst implantation in early embryo development.

Habitual abortions in 678 healthy patients: investigation and prevention.

The objective of this study of patients with habitual abortion (HA), was to determine their autoimmune profile and to try to prevent new abortions using low-dose aspirin for 7 months with prednisone in the first trimester only, or with low-dose aspirin alone. A total of 678 healthy patients with three or more HA were investigated for antiphospholipid antibodies, antinuclear and antithyroid antibodies. Among these patients, 277 pregnant women were treated, 214 were given prednisone and aspirin (161 autoantibody-negative and 53 autoantibody-positive women), and 63 autoantibody-negative women received aspirin alone. Autoantibodies were present in 33.9% of the patients, in 82.6% of them anticardiolipin antibodies were found to be isolated or associated with antiprothrombin, antithyroid, circulating anticoagulant, antinuclear or anti-beta2 glycoprotein 1 antibodies. In autoantibody-negative pregnant women treated by prednisone and aspirin or aspirin alone, the success rate of live births was 90.7% (146 out of 161) and 74.6% (47 out of 63) respectively (P < 0.01). In autoantibody-positive patients treated with prednisone and aspirin the success rate was 84.9% (45 out of 53) (not significant). Prednisone and aspirin seemed to be as efficient in autoantibody-negative or positive women but better than aspirin alone in autoantibody-negative women. A double-blind trial is in progress to confirm these results.

[Embryotoxic factors and recurrent spontaneous abortions]. / Facteur embryotoxique et interruptions spontanées répétées de grossesse.

About 30% of recurrent spontaneous abortions remain unexplained by traditional or biological anomalies. The purpose of this work was to investigate embryotoxic factors produced by trophoblast stimulated lymphocytes from women with unexplained recurrent spontaneous abortion. The samples from 36 women with recurrent abortion before and during the next pregnancy and from 7 women with normal pregnancies and no history of spontaneous abortion have been tested. The lymphocytes were stimulated with trophoblastic extracts. The supernatants of the stimulated lymphocytes were tested in a 4-cell mouse embryo culture. The secretion of embryotoxic factor was determined if the number of life blastocysts was less than 50% of control values after 4 days. The lymphocytes from 59% women with 3 or more recurrent abortion produced the embryotoxic factor, this factor may be useful in predicting pregnancy outcome in women with a history of unexplained recurrent miscarriage. The embryotoxic factor might be a new cause of recurrent abortion and a predictive factor.

Human auto-anti-idiotypic antibodies to mite-specific IgE can degranulate human basophils in vitro.

BACKGROUND: Anti-idiotypic antibodies (anti-Ids) to specific IgE antibodies are formed spontaneously during an anti-allergen immune response and can be induced by immunotherapy. Although anti-Ids can down-regulate the production of IgE antibodies, at least in experimental models, their possible role in the modulation of target cell reactivity remains ill-defined. OBJECTIVE: The capacity of human anti-Ids to modulate the release of histamine was examined in an in vitro system of human basophil degranulation. Anti-Ids were prepared from the serum of six Dermatophagoides pteronyssinus (Dp)-hypersensitive patients suffering from atopic dermatitis and who had never been desensitized. Basophils were obtained from the blood of atopic donors. The extent of histamine release was determined using a fluorometric assay. RESULTS: We show that: anti-Ids trigger the release of histamine in an allergen-specific, dose- and IgE-dependent manner; the release is not due to the presence of allergen and/or anti-IgE antibodies; and that the degranulating activity can be removed by absorption with affinity-purified anti-Dp antibodies of the corresponding patient. CONCLUSION: These results indicate that spontaneously produced human anti-Ids can modulate the reactivity of human basophils.

Binding of serum autoantibodies to sialidase-treated tracheal epithelial cells. Determination of autoantibodies isotypes in normal and influenza virus infected guinea pig sera.

Cultured epithelial cells isolated from guinea pig trachea were treated with Vibrio cholerae sialidase. The treatment was not cytotoxic and resulted in membrane desialylation as assessed by measurement of sialic acids released, along with an increased fixation of the galactose-specific lectin peanut agglutinin. After incubation in serum from normal guinea pigs, membrane-bound immunoglobulins were detected using peroxidase-labelled antibodies. Sialidase-treated cells bound significantly more IgM than controls (P < 0.0005), whereas binding of IgG was not significantly different between treated and untreated cells (0.1 < P < 0.375); IgA were never detected. In influenza-infected guinea-pigs, as assessed by reactivity with peanut agglutinin, the tracheal and lung epithelium, as well as alveolar cells were hyposialylated. In these animals, the level of serum IgG autoantibodies capable to bind sialidase treated cultured cells increased, while the level of IgM autoantibodies did not change. These autoantibodies may participate in cellular dysfunctions and modified bronchoreactivity that occur during infection of the respiratory tract by sialidase-producing microorganisms, either through activation of the complement system, or subsequently to their reaction with cells expressing membrane complement and/or Fc receptors.

No evidence for the expression of the progesterone receptor on peripheral blood lymphocytes during pregnancy.

The expression of the progesterone receptor in human peripheral blood lymphocytes was analysed, using an enzyme linked immunosorbent assay (Abbott PgR-EIA monoclonal), in order to evaluate its prognostic character in the context of spontaneous abortion. Cytosols were prepared from lymphocytes of 24 healthy pregnant women (11 first, 10 second and three third trimester), seven healthy non-pregnant women, nine women with recurrent spontaneous abortion, and six healthy men. In addition, a human breast carcinoma cell line (ZR-75-1), which expresses the progesterone receptor, was analysed throughout. The ZR-75-1 cell line showed an expression of 642 fmol/mg whereas lymphocytes of pregnant women showed an expression < or = 4 fmol/mg. Lymphocytes of non-pregnant women, women with threatened pre-term delivery, and men showed equivalent levels: 3 +/- 1, 3 +/- 2 and 5 +/- 4 fmol/mg respectively. These results show that there is no evidence of specific expression of the progesterone receptor in pregnancy and exclude any prognostic character in spontaneous abortion. A role for the progesterone receptor in the mechanism of the known effect of progesterone on peripheral blood lymphocytes is also excluded.

HLA antigen-sharing in couples with repeated spontaneous abortions and the birthweight of babies in successful pregnancies.

Previously, several groups reported an increase in HLA antigen-sharing in couples suffering from unexplained repeated spontaneous abortions. It was felt necessary to find out if HLA sharing could have any effect on children born after a successful pregnancy. The birthweight figures of children of 76 couples with repeated spontaneous abortions were analyzed. The results show a significantly lower birthweight in babies born from those couples, presenting a high incidence of HLA antigen-sharing, particularly concerning class II antigens.

HLA-DRw52a is involved in alloimmunization against PL-A1 antigen.

The alloimmunization against the platelet PL-A1 antigen is strongly associated with a HLA class II structure in mothers of thrombocytopenic neonates. Most of the immunized women have first been shown to possess the DR3 specificity and subsequently the DRw52 allele. The 18 immunized mothers studied here by restriction fragment length polymorphism analysis had the DRw52a specificity at the DRB3 locus whatever their HLA-DRB1 gene product. This finding strongly suggests that the DRB3 chain is directly involved in the presentation of the PL-A1 antigen to the specific T cell. In addition, the similarities between DR3 and DRw52 structures due to a hypothetical gene conversion event should be considered in order to understand the high frequency of DR3 among the DRw52a-responding women. Alternatively, the high frequency of DR3 among the DRw52-responding mothers might be due to the high responder status associated with the former specificity.

Lymphocytic progesterone receptors in normal and pathological human pregnancy.

The progesterone receptor-specific monoclonal antibody (MoAb) mPRI was tested for its reactivity towards peripheral blood lymphocytes (PBL) of 49 healthy pregnant women, nine pregnant women with clinical symptoms of threatened preterm delivery, seven women with recurrent spontaneous abortion, ten women in labour and ten women with spontaneous abortion. Lymphocytes of 12 healthy age-matched non-pregnant volunteers were used as controls. Lymphocytes of nine healthy pregnant women at the 1st trimester of pregnancy and those of two non-pregnant donors were tested for the presence of estrogen and progesterone receptors by enzyme immunoassay. PBL of healthy pregnant women contained significantly more positive cells than those of non-pregnant controls. Furthermore, the number of receptor-containing cells increased in parallel with gestational age. In blood samples drawn during labour, as well as in those obtained from women with spontaneous abortion or clinical symptoms of threatened pre-term delivery, the percentage of positively stained lymphocytes was significantly lower than normal pregnancy values. This was also the case in peripheral blood of pregnant women with a history of recurrent spontaneous abortions.

Enhancement of the histamine releasing activity of mouse monoclonal anti-human IgE antibody xb6-16 when present in aggregated or complexed forms.

FPLC purification of mouse monoclonal anti-human IgE antibody xb6-16 showed 2 major peaks of different molecular weight, peak 1 (greater than 10(6) d) and peak 3 (1.6 x 10(5) d). Peak 1 consisted of IgG1 and IgM, peak 3 of IgG1 only. On a protein weight basis, peak 1 was 100 times more potent than peak 3 in inducing histamine release from human basophils. Preincubation of peak 3 with anti-IgG1 enhanced the mediator release triggered by this fraction. On this basis, the potentiating effect of aggregated IgG1 or IgG1-IgM complexes on mediator release from basophils is discussed.

In vitro inhibition of the classical and alternate pathways of activation of human complement by N acetyl aspartyl glutamic acid (NAAGA).

A synthetic dipeptide, magnesium salt of N-acetyl-L-aspartyl-glutamic acid (NAAGA) identical to a natural dipeptide found as traces in cerebral tissues of mammalian brains, was shown to inhibit, in vitro, the hemolytic activity of both classical and alternate pathways complement; the required concentration of NAAGA was 2 to 10 mM. Cross immuno electrophoretic analysis demonstrated an inhibition of C3 cleavage by both classical and alternate pathway C3 convertases with 24 mM NAAGA. As expected, if C3 convertases were really the target of inhibition, the release of highly inflammatory C3a, C5a fragments scored by R.I.A. was impaired when complement was incubated with activators of classical and alternate pathways. Such a low molecular weight dipeptide, quite atoxic and inhibiting the complement dependent cytotoxicity and release of phlogistic by-products could be interesting for pharmacological manipulation of complement activation in inflammatory processes.

Management of alloimmune neonatal and antenatal thrombocytopenia.

Neonatal and antenatal alloimmune thrombocytopenia is induced by maternal antibodies against platelet-specific fetal antigens. This disease is rare but potentially severe because of intracranial bleedings which may occur during pregnancy or around birth. In the last decade our knowledge of this disorder has markedly advanced. New techniques are used in platelet immunology. New platelet antigens involved in these perinatal thrombocytopenias have recently been discovered. A group of women likely to produce the responsible platelet antibodies has been genetically defined as regards the PLA1 antigen. The quality of the sonographies and the possibility of performing cord vein puncture in early pregnancy afford a new approach in the management of perinatal alloimmune thrombocytopenias. But more must be done to prevent the complications of this disease.

[Abortions of immunologic origin]. / Avortements d'origine immunologique.

The immunology of pregnancy involves a series of systemic and above all local events, at the feto-maternal interface. These immunological events may explain the paradoxical non-rejection and development of the allogeneic conceptus within of the maternal body. The immunodeviation of the maternal immune system towards fetal tolerance may be altered or insufficient leading to true abortive diseases. The alteration of this tolerance may be the result of auto-immune abnormalities, particularly when autoantibodies are discovered in woman's serum. These antibodies are the antithromboplastin, antiphospholipid and antinuclear antibodies. They can be responsible for abortion even if the clinical symptoms of the lupus disease are absent. Abortive events occur at all stages of pregnancy. Table 1 shows that abortions with autoantibodies are more frequent when the accident occurs in the late stage of pregnancy. The therapy with corticoids and aspirin will be modulated in connection with the results. The second immune etiology is an insufficient production of immunological events usually involved in normal pregnancies. The current means available to detect this incompetency are imperfect because only systemic factors may be explored when local events are the most involved. We found a good correlation, in these women with recurrent early abortions of unknown etiology, between the production of anti-husband lymphocytes (AAP) and the success of a subsequent pregnancy. Women who suffer from recurrent spontaneous abortions of unknown etiology, without autoimmune abnormalities and without antipaternal antibodies (AAP), may profit from a therapy using the husband's leucocyte injections, which allows them to give birth to a normal child in 85% of the cases, whereas without treatment the success rate is only 37% of the pregnancies. When this therapy is applied in accurate conditions, its inocuousness seems well established. Another kind of this immunomodulator therapy has been reported: it uses unrelated donor leucocytes. Its efficiency seems to be similar to that of the husband's leucocyte injections.