The protective effect of berberine against lipopolysaccharide-induced abortion by modulation of inflammatory/immune responses.

OBJECTIVE: Berberine is an isoquinoline derivative alkaloid with anti-inflammatory activity. In this study, we investigated the protective effects of berberine in prevention of LPS-induced abortion. MATERIALS AND METHODS: On the gestation day (GD) 9.5, the pregnant mice were injected with low, medium, and high doses of berberine or with PBS. After 4 h, berberine or PBS-pretreated mice were injected with LPS. On GD 11.5, blood samples and uterine tissues were collected from treated mice and percentage of abortion and serum levels of NO, TNF-α, IL-10, and IL12p70 were measured by macroscopic examination and sandwich ELISA, respectively. RESULTS: Our findings show that mice injected with berberine were resistant to LPS-induced abortion. We also found that this treatment prevents the reduction of IL-10 and the enhancement of NO, TNF-α, and IL-12p70 in LPS-treated pregnant mice. CONCLUSIONS: Taken together, our results suggest that berberine as an anti-inflammatory agent has protective effects on LPS-induced abortion by modulation of inflammatory/immune responses.

Bovine Neonatal Monocytes Display Phenotypic Differences Compared With Adults After Challenge With the Infectious Abortifacient Agent Neospora caninum.

The neonatal period represents a window of susceptibility for ruminants given the abundance of infectious challenges in their environment. Maternal transfer of immunity does not occur in utero but post-parturition, however this does not compensate for potential deficits in the cellular compartment. Here we present a cellular and transcriptomic study to investigate if there is an age-related difference in the monocyte response in cattle during intra-cellular protozoan infection. We utilized Neospora caninum, an obligate intracellular protozoan parasite that causes abortion and negative economic impacts in cattle worldwide, to study these responses. We found neonatal animals had a significant greater percentage of CD14+ monocytes with higher CD80 cell surface expression. Adult monocytes harbored more parasites compared to neonatal monocytes; additionally greater secretion of IL-1ß was observed in neonates. Microarray analysis revealed neonates have 535 genes significantly upregulated compared to adult with 23 upregulated genes. Biological pathways involved in immune response were evaluated and both age groups showed changes in the upregulation of tyrosine phosphorylation of STAT protein and JAK-STAT cascade pathways. However, the extent to which these pathways were upregulated in neonates was much greater. Our findings suggest that neonates are more resistant to cellular invasion with protozoan parasites and that the magnitude of the responses is related to significant changes in the JAK-STAT network.

Proteomic identification of immunodominant membrane-related antigens in Campylobacter jejuni associated with sheep abortion.

Campylobacter jejuni clone SA is the predominant agent inducing sheep abortion and a zoonotic agent causing gastroenteritis in humans in the United States. In an attempt to identify antigens of clone SA that may be useful for vaccine development, immunoproteomic analyses were conducted to characterize the membrane proteome of C. jejuni clone SA. 2-DE of C. jejuni membrane-related proteins was followed by immunoblotting analyses using convalescent sera that were derived from ewes naturally infected by C. jejuni clone SA. Totally 140 immunoreactive spots were identified, 50 of which were shared by all tested convalescent sheep sera. Conserved and immunodominant spots were identified by mass spectrometry. Among the 26 identified immunogenic proteins, there were 8 cytoplasmic proteins, 2 cytoplasmic membrane proteins, 11 periplasmic proteins, 3 outer membrane proteins, and 2 extracellular proteins. Notably, many of the immunodominant antigens were periplasmic proteins including HtrA, ZnuA, CjaA, LivK, CgpA, and others, some of which were previously shown to induce protective immunity. Interestingly, 11 immunoreactive proteins including 9 periplasmic proteins are known N-linked glycosylated proteins. These findings reveal immunogens that may potentially elicit protective immune responses and provide a foundation for developing vaccines against C. jejuni induced sheep abortion. BIOLOGICAL SIGNIFICANCE: Campylobacter jejuni clone SA is the predominant agent inducing sheep abortion and incurs a significant economic loss to sheep producers. This emergent strain is also a zoonotic agent, causing gastroenteritis in humans. However, the immunogens of C. jejuni induced abortion are largely unknown. Considering the significance of C. jejuni clone SA in causing sheep abortion and foodborne illnesses, protective vaccines are needed to control its transmission and spread. Additionally, immunological markers are required for detection and identification of this highly pathogenic clone. To address these needs, we applied an immunoproteomic approach to identify the membrane-associated antigens of this highly virulent C. jejuni clone associated with sheep abortions in the U.S. The findings reveal immunogens that may potentially elicit protective immune responses and provide a foundation for developing vaccines against C. jejuni induced sheep abortion.

Azithromycin prevents pregnancy loss: reducing the level of tumor necrosis factor-alpha and raising the level of interleukin-10 in rats.

The aim of this study was to determine the effect of azithromycin on LPS-induced pregnancy loss. Thirty-six pregnant female Wistar rats were divided into 4 equal groups as follows: control group, where 0.3 mL of normal saline solution was administered intravenously on day 10 of pregnancy; azithromycin group, where azithromycin was administered orally at 350 mg kg(-1) day on days 9, 10, and 11 of pregnancy; lipopolysaccharide group, where LPS was administered intravenously via the tail vein at 160 µg kg(-1) on day 10 of pregnancy; and the azithromycin + LPS group, where azithromycin was administered orally at 350 mg kg(-1) day on days 9, 10, and 11 of pregnancy and LPS was administered intravenously at 160 µg kg(-1) on day 10 of pregnancy. Blood samples were obtained from the tail vein on day 10 of the experiment. Pregnancy rates were determined. Tumor necrosis factor-alpha (TNF- α ) and interleukin (IL-10) levels were measured by ELISA. Azithromycin prevented (P < 0.05) LPS-induced pregnancy loss. Higher TNF- α and IL-10 levels were measured (P < 0.05) in the LPS and azithromycin + LPS groups, respectively. In conclusion, azithromycin may be useful in infection- or endotoxemia-dependent pregnancy loss.

IFN-γ expression in placenta is associated to resistance to Chlamydia abortus after intragastric infection.

Intragastric infection mimics the natural route of infection of Chlamydia abortus (etiological agent of ovine enzootic abortion). In the mouse model, intragastric experimental infection induces very mild signs of infection followed by late term abortions, as it is shown by the natural ovine host. In order to evaluate the immune mechanisms associated to the dissemination of the pathogen from the gastrointestinal tract, we have administered an intragastric dose of C. abortus to pregnant mice. Systemic and local expression of cytokines, tissue colonization and excretion of bacteria after parturition were monitored during pregnancy. Susceptible CBA/J mice showed a higher bacterial colonization of the placenta and excretion of live bacteria after parturition that were related to a higher local IL-10 expression. By contrast, resistant C57BL/6 mouse strain had higher local IFN-γ mRNA expression in the placenta just before parturition and a transient bacterial colonization of the reproductive tract, with no excretion of C. abortus after parturition. In summary, intragastric infection not only mimics the natural route of infection of C. abortus, but can also be useful in order to understand the immunopathogenesis of chlamydial abortion in the mouse.

Pathogens and the placental fortress.

Placental infections are major causes of maternal and fetal disease. This review introduces a new paradigm for placental infections based on current knowledge of placental defenses and how this barrier can be breached. Transmission of pathogens from mother to fetus can occur at two sites of direct contact between maternal cells and specialized fetal cells (trophoblasts) in the human placenta: firstly, maternal immune and endothelial cells juxtaposed to extravillous trophoblasts in the uterine implantation site and secondly, maternal blood surrounding the syncytiotrophoblast (SYN). Recent findings suggest that the primary vulnerability is in the implantation site. We explore evidence that the placental SYN evolved as a defense against pathogens, and that inflammation-mediated spontaneous abortion may benefit mother and pathogen.

Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus.

Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.

Inflammatory agents involved in septic miscarriage.

Even though the understanding of the cause of early pregnancy loss due to chromosomal abnormalities has improved, there is a dearth of knowledge of the causes of loss in euploid conceptuses. Maternal infections are a cause of abort in humans, but the mechanisms are not clear, so we have developed a murine model to study the mechanism of septic abortion by inducing embryonic resorption (ER) with lipopolysaccharide (LPS). We demonstrated that augmented production of nitric oxide (NO) and prostaglandins (PG) is involved in ER, and that inhibitors of their synthesis could prevent ER. Also, we observed an increase in the oxidative damage, evidenced by nitration of tyrosine proteins, due to the peroxynitrite anion. Since an association between chronic marijuana smoking and early miscarriage has been shown in women, we studied the participation of anandamide (AEA), the principal endocannabinoid, on the mechanism of action of LPS. We showed that LPS-induced NO synthesis and tissue damage were mediated by AEA, and that this endotoxin inhibited AEA degradation and increased its synthesis. These results suggest that several inflammatory molecules participate in the mechanism of early pregnancy loss and that their modulation could be useful tools to prevent it.

Endocannabinoid system and nitric oxide are involved in the deleterious effects of lipopolysaccharide on murine decidua.

Endocannabinoids are an important family of lipid-signaling molecules that are widely distributed in mammalian tissues and anandamide (AEA) was the first member identified. The uterus contains the highest concentrations of AEA yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. Previous results from our laboratory have shown that AEA modulated NO synthesis in rat placenta. The production of small amounts of nitric oxide regulates various physiological reproductive processes such as implantation, decidualization and myometrial relaxation. But in an inflammatory setting such as sepsis, NO is produced in big amounts and has toxic effects as it is a free radical. The results presented in this study indicate that LPS-induced NO synthesis and tissue damage were mediated by AEA. Decidual LPS-induced NO production was abrogated either by co-incubation with CB1 (AM251) or CB2 (SR144528) antagonists which suggests that both receptors could be mediating this effect. On the other hand, LPS-induced tissue damage and this deleterious effect was partially abrogated by incubating tissue explants with LPS plus CB1 receptor antagonist. Our findings suggest that AEA, probably by increasing NO synthesis, participates in the deleterious effect of LPS in implantation sites. These effects could be involved in pathological reproductive events such as septic abortion.

Escheriosome-mediated delivery of recombinant ribosomal L7/L12 protein confers protection against murine brucellosis.

Brucellosis is an important zoonotic disease that causes abortion in cattle and undulant fever, arthritis, endocarditis and meningitis in human. In spite of the fact that immunization could be an efficient measure to control brucellosis, not a single ideal vaccine against this important disease has been developed so far. In order to develop an effective vaccine against Brucella abortus (B. abortus), various protective immunodominant gene/protein products of the pathogen have been studied in combination with different adjuvants. For example, recombinant ribosomal protein L7/L12 (rL7/L12) although an interesting T-cell antigen, normally failed to evoke protective immune response when used in free form. In the present study we have demonstrated that Escherischia coli (E. coli) lipid liposome (escheriosome)-mediated cytosolic delivery of recombinant rL7/L12 protein can elicit strong immunological responses in the Balb/c mice. In contrast, egg PC/Chol liposome entrapped rL7/L12, in a manner similar to its free form, was found to impart relatively poor immune response. Furthermore, escheriosome entrapped rL7/L12 protein elicited high IgG2a isotype response suggestive of its relevance in imparting protection against brucellosis in mice. Altogether the present study is a clear indicative of the possible use of escheriosome-based delivery of rL7/L12 protein to induce protective immune responses against experimental murine brucellosis.

Evaluation levels of cytokines in amniotic fluid of women with intrauterine infection in the early second trimester.

Amniotic fluid was obtained from 180 patients by amniocentesis at 16-22 weeks of gestation and assayed for the levels of interleukin (IL)-6, IL-8, leukocyte elastase (LE), and glucose. Ten of cases had clinical symptoms, such as uterine contraction, genital bleeding, and cervical ripening, and the other 170 were assessed for fetal chromosomal features. Four of the ten cases with uterine contraction developed abortion, while 10 of those screened had findings of fetal chromosomal anomalies, and 7 cases then underwent induced abortion artificially. In the cases of abortion, levels of IL-6, IL-8 and LE were higher than in the samples from the 160 pregnant women without clinical symptoms and a normal karyotype, while glucose in amniotic fluid was lower. Of 6 cases with clinical symptoms, but not developing abortion, 4 developed preterm labor, and in these IL-6 and IL-8 also were significantly elevated, with LE being slight high compared to normal. The results suggest that IL-6, IL-8, LE, and glucose in amniotic fluid at early second trimester can be used as markers of severe infection in the uterus, and with the first two being particularly sensitive.

A 'minimum dose' of lipopolysaccharide required for implantation failure: assessment of its effect on the maternal reproductive organs and interleukin-1alpha expression in the mouse.

Genital tract infections caused by gram-negative bacteria induce abortion and are one of the most common complications of human pregnancy. This study was carried out to decipher the mechanism of gram-negative bacterial lipopolysaccharide (LPS)-induced pregnancy loss, using a mouse (Park strain) model. Since many of the biological effects of LPS are mediated by interleukin (IL)-1alpha, the role of IL-1alpha in LPS-induced pregnancy loss was studied. Pregnant female animals were injected intra-peritoneally (i.p.) with different doses (1 to 50 microg) of LPS from Salmonella minnesota Re-595, on day 0.5 of pregnancy. We found that 250 microg/kg body weight (i.e. 5 microg/female mouse) of LPS when given on day 0.5 of pregnancy was the 'minimum dose' (MD) required to completely inhibit the implantation of the blastocyst in the mouse. The effect of this dose on the pathophysiology of the various reproductive organs (i.e. uterus, ectoplacental cones, developing fetus, ovaries etc.) was assessed on day 14 of pregnancy. The effects of this dose on the level and pattern of expression of the proinflammatory cytokine IL-1alpha in the maternal uterine horns and preimplantation stage embryos were studied by RT-PCR. A single dose (100 ng/mouse) of recombinant mouse IL-1alpha was given i.p. to pregnant females on day 1 of pregnancy to study its effect on implantation. Our results show that treatment of the pregnant animals with LPS may alter cell proliferation and induce leukocyte infiltration, degeneration of luminal glandular epithelium, and hyperplasia in the various reproductive organs, and may also alter both embryonic and uterine IL-1alpha expression. IL-1alpha administration also caused implantation failure similar to that of LPS. The observations suggest that the determined MD of LPS may alter the expression of developmentally important proinflammatory cytokines such as IL-1alpha, which could, in turn, inhibit the normal processes of blastocyst implantation. Therefore, it is proposed that the LPS-induced histopathological alterations in the various reproductive organs of pregnant animals could be mediated by IL-1alpha and this may be one of the causes of failure of blastocyst implantation in the mouse.

Immune regulation during pregnancy and host-pathogen interactions in infectious abortion.

The immunological mechanisms that govern the success of pregnancy in outbred mammals are complex. During placental formation the invasion of fetal cells into maternal tissue must be controlled to prevent damage to the mother. Equally, maternal recognition of pregnancy must be such that allorejection of the fetus does not occur. Despite the complexity of this phenomenon, it is clear that cytokines play a crucial role at the maternofetal interface and in the periphery to ensure that pregnancy proceeds successfully. Inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) can exert detrimental effects in the placenta and tend to be present at low concentrations, whereas the regulatory cytokines interleukin (IL)-10 and tranforming growth factor-beta (TGF-beta) are beneficial and tend to predominate. This means that infection with pathogens that target the placenta and that elicit inflammatory responses may cause abortion by giving rise to a detrimental combination of cytokines that causes damage but does not control the disease. Infectious abortion is discussed in the context of the modulation of host immune responses during pregnancy, taking into account the different placental structures present in human beings, rodents and ruminants.

[Procalcitonin as monocytic marker for early diagnosis in septic abortion]. / Procalcitonin als monozytärer Marker für die Frühdiagnostik bei septischem Abort.

BACKGROUND: In search of sensitive and specific markers of systemic infection procalcitonin (PCT) recently was promoted to the focus of clinical research. Little is known about the biology of PCT and until now no data have been presented about clinical importance of PCT in obstetric patients. PATIENT AND METHODS: Daily PCT values in a 17 year old patient with septic abortion were compared with established markers of systemic inflammation. Cultivated monocytes were analyzed by the means of indirect immunofluorescence for intracellular distribution of PCT. Additionally, PCT release into culture medium was examined. RESULTS: PCT values in comparison with established inflammation markers was demonstrated in the patient with septic abortion. Indirect immunofluorescence studies revealed the presence of PCT within monocytes. In the supernatants of monocyte cultures PCT was detectable under control conditions. Stimulation with lipopolysaccharide resulted in the increased PCT concentrations both in the supernatants of healthy and patient monocyte cultures. CONCLUSIONS: In the given patient PCT was superior to other inflammation markers with regard to early and progression diagnosis. Peripheral blood monocytes appear to be a potential site of inflammation-induced PCT production. For the first time intracellular distribution pattern and release of PCT from human monocytes was described. DISCUSSION: Based on the presented data broad clinical studies devoted to PCT evaluation in obstetric patients seem to be promising. As till now the interpretation of increased PCT values depended on empirical knowledge, extensive studies of the potential production site as well as its biological significance should be performed, too.

Comparison of five tests for the detection of antibodies against chlamydial (enzootic) abortion of ewes.

Five tests for antibodies against chlamydial (enzootic) abortion of ewes were compared using 255 sera from experimentally (group 1) or naturally (group 2) infected animals, flocks free of the disease (group 3) and individual animals testing positively by the complement fixation test but from flocks with no evidence of chlamydial abortion (group 4). Sera from five specific pathogen-free lambs vaccinated with two different subtypes of Chlamydia pecorum were also included (group 5). All tests used some form of processed culture of C psitiaci as antigen. Specificities, established with group 3 and 4 sera, ranged between 96 per cent (ELISA using lipopolysaccharide antigen) and 59 per cent (Immunocomb). Reactions with group 5 sera suggested that the cause of false positive results in the field might be cross-reactive antibodies against the arthritogenic subtype of C pecorum. Sensitivities, established with groups 1 and 2 sera, ranged between 81 per cent (Immunocomb) and 51 per cent (ELISA using solubilised protein antigen). The minimum sample sizes required to be 95 per cent certain of detecting at least five seropositives in two infected flocks (combined data) were 15 to 48, dependent on the test applied. The Western blot test, applied to a proportion of samples, yielded no false positives with group 3 sera but 31.7 per cent with group 4 sera. Thus, none of the tests in this comparison emerged as sufficiently satisfactory in all respects, suggesting that further improvements in chlamydial serology must come through the use of non-native antigens or in the form of a competitive ELISA.

Identification of Ehrlichia risticii as the causative agent of two equine abortions following natural maternal infection.

Two pregnant mares diagnosed as having equine monocytic ehrlichiosis based on history, clinical signs, and high serum antibody titers to Ehrlichia risticii aborted subsequent to recovery from illness. Mare 1 and mare 2 experienced clinical illness at 120 and 143 days of gestation and aborted at 203 and 226 days of gestation, respectively. The fetuses were expelled in fresh condition, and both mares retained their placentas upon abortion. Gross findings for the fetuses included meconium staining and petechiation of external surfaces. Internally, there was increased volume of feces within the small and large intestines and liver discoloration with enlargement. Microscopic findings included lymphohistiocytic enterocolitis, hepatitis, and myocarditis. Lymphoid hyperplasia and depletion were present in spleen, thymus, and lymph nodes. Ehrlichia risticii was recovered from bone marrow, spleen, lymph node, colon, and liver of the first fetus and bone marrow and colon of the second fetus. Electron microscopic evaluation of the organism isolated in cell culture revealed morphology consistent with E. risticii. The isolated organism was inoculated into a naive pony, and this pony developed high levels of antibody against E. risticii, became ehrlichemic, and developed clinical signs of depression, anorexia, and mild diarrhea. These findings confirm that E. risticii is an abortifacient under conditions of natural infection and should be considered as a differential diagnosis of equine abortions.

[A skin allergy test in chlamydial abortion in sheep]. / Kozný alergický test pri chlamýdiovom potrate oviec.

46 head of pregnant sheep, the Tsigaya breed, were subjected to the skin allergy test and subsequently divided into two groups. Sheep were in the 3rd month of gravidity and were a part of a flock consisting of 300 head, in which chlamydia-induced abortion was recorded in sheep. The skin allergic test was done by the Rodolakis et al. (1977) method, modified by us, to indicate the level of cell-mediated immune response. Simultaneously with it, serological examinations (complement fixation test--CFT) were performed to find out the levels of antibody against Ch. psittaci. The results of skin allergy test (SAT) and serological examination in sheep after bivalent vaccine administration are given in Tab. II. Of the total number of sheep ranked to vaccinated group, 18 head responded positively on SAT. After vaccination, 12 head responded positively though previously responded negatively. In vaccinated group one abort recorded in the sheep. No. 12 which was on the 0 day slightly positive in the skin test. High levels of antibody were found after abortion and the skin test was highly positive. The results of SAT and serological examination in sheep, when placebo was administered, are given in Tab. III. 6 sheep aborted in the group, placebo was administered, are given in Tab. III. 6 sheep aborted in the group, of which 5 were negative and one was slightly positive on the day 0 in SAT. In 4 sheep abort was accompanied with significant increase in humoral antibody against Ch. psittaci. In sheep which aborted and were negative in SAT on the day 0, a marked positivity has been indicated in the replicated SAT test.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of specific trophoblastic beta 1-glycoprotein on changes in the cellular link of immunity in infected abortion]. / Vliianie spetsificheskogo trofoblasticheskogo beta 1-glikoproteida na izmenenie kletochnogo zvena immuniteta pri infitsirovannom aborte.

Effects of specific trophoblastic beta-1-glycoprotein (TBG) on T- and B-cell immunity have been explored in 107 patients with purulent septic complications of abortion. TBG showed a marked suppressor effect on T lymphocytes. Persistence of TBG in blood of patients with infection complicated abortion maintains immunodeficient states. The presence of TBG in serum may increase the risk of severe septic complications of illegal abortion.

PIP: The immunosuppressive activity of trophoblastic beta 1-glycoprotein (TBG) was studied in 107 patients with septic complications of induced abortion. The patients were divided into 2 groups. Group 1 consisted of 46 women with local septic complications, and Group 2 included 61 women with generalized complications. Healthy pregnant women were used as controls. The patients were examined immediately after admission to the hospital and 5-8 days later. The functional state of cellular immunity was evaluated by the county of T- and B-lymphocytes in the peripheral blood, by lymphocyte transformation induced by phytohemagglutinin, and by the index of lymphocyte stimulation. Inhibition of cellular immunity in patients with septic complications was found to depend on the presence of TBG in the peripheral blood. In patients in Group 1, total lymphocyte count was significantly lower in the patients with the presence of TBG, compared with that in the patients in whom TBG was absent (0.53x10(9) and 1.06x10(9), respectively). Similar decline was observed in patients with generalized septic complications (Group 2). In patients with septic complications of induced abortion regardless of their severity, the indices of reaction of spontaneous blast transformation were close to those in healthy pregnancy women only in an absence of TBG. The patients with circulating TBG showed inhibition of reaction of blast transformation. The index of lymphocyte stimulation was also inhibited in the presence of TBG (32.8 in Group 1, 22.0 in Group 2 and 60.0 in health pregnant women; in the absence of TBG, the index of lymphocyte stimulation in Groups 1 and 2 was 42.1 and 28.8, respectively.