IFPA Meeting 2010 Workshops Report II: Placental pathology; trophoblast invasion; fetal sex; parasites and the placenta; decidua and embryonic or fetal loss; trophoblast differentiation and syncytialisation.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb.4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells.

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.

Evaluation of hypersensitivity to microencapsulated ampicillin in guinea pigs.

The purpose of this study was to determine if the sustained release of ampicillin from a biodegradable drug-delivery system (microencapsulated ampicillin anhydrate (MEAA)) will increase or decrease the intensity of a hypersensitivity reaction compared with that observed with free drug. Ovalbumin, which is known to elicit a marked hypersensitivity reaction in guinea pigs, and microencapsulated ovalbumin (MOVA) were tested in parallel with ampicillin and MEAA. Guinea pigs were sensitized biweekly by subcutaneous and intramuscular injections of ampicillin, MEAA, ovalbumin, MOVA or placebo microspheres (test articles), each mixed with Freund's adjuvant, and challenged 2 weeks later, intradermally, with the free compounds. In a separate set of experiments, guinea pigs were sensitized by implantation of the same agents in the caudal thigh of anaesthetized animals. Skin allergic reactions were tested at 1 and 3 weeks following local implantation of the test articles. Sera of sensitized guinea pigs were tested for specific IgG antibodies by enzyme-linked immunosorbent assay, and skin samples from the site of the inflammatory reaction were fixed, stained and evaluated histologically. Guinea pigs sensitized systemically with MEAA or MOVA showed smaller, but not statistically different skin allergic response than animals given corresponding free compounds. However, guinea pigs sensitized by local implantation of MEAA showed a significantly lower inflammatory response (P < 0.0001) than those given an equivalent dose of the free drug. Guinea pigs sensitized with placebo microspheres showed a low inflammatory skin reaction which was similar to those sensitized with all doses of MEAA. There was no significant difference in specific IgG antibody response in the sera of guinea pigs sensitized locally with either free or microencapsulated ampicillin or ovalbumin. Histology of skin revealed a milder inflammatory reaction with MEAA or MOVA than with ampicillin or ovalbumin, respectively. We conclude that the encapsulated ampicillin or ovalbumin and subsequent release of each agent will elicit a reduced hypersensitivity reaction in guinea pigs than will the free agent.

Effect of microencapsulated ampicillin on cell-mediated immune responses in mice.

The effects of free ampicillin, microencapsulated ampicillin anhydrate (MEAA) and antibiotic-free microspheres on the cell-mediated immune response in Balb/c mice were measured by lymphoproliferation assay, delayed-type hypersensitivity (DTH) and cytokine production. Injection into mice for seven consecutive days with equivalent subcutaneous doses of ampicillin, MEAA or placebo microspheres did not produce any consistent change in lymphocyte proliferation nor did it affect DTH responses or interleukin-2 production. Although the production of interleukin-4 in mice treated with ampicillin or MEAA increased compared with the control mice, this increase was not statistically significant. These results indicate that ampicillin and MEAA have similar effects on cell-mediated immunity in mice.

Development of chronic mandibular osteomyelitis in a miniswine model.

Previous attempts to develop a reproducible model of chronic mandibular osteomyelitis have met with limited success. In this study, osteomyelitis was produced in the mandibles of eight adult Yucatan miniswine by the intramedullary application of sodium morrhuate, Staphylococcus aureus, and either polymethylmethacrylate bone cement or bone wax. At 8 weeks' postinfection, the mandibles were surgically debrided and specimens were obtained for culture. Although all of the animals developed clinical evidence of osteomyelitis that was supported by positive cultures, the original organism (S aureus) was recovered only from those animals where bone wax had been used to seal the cortical defects. This animal model may be useful for evaluating newer treatment modalities for chronic osteomyelitis.

Detection of Schistosoma mansoni circulating cathodic antigen for evaluation of resistance induced by irradiated cercariae.

The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans.

Diagnosis of human schistosomiasis by detection of circulating cathodic antigen with a monoclonal antibody.

Monoclonal antibody (MAb) 5H11 reacted with repeating epitopes on Schistosoma mansoni circulating cathodic antigen (CCA) and detected CCA in sera of Egyptian S. mansoni-infected patients. MAb 5H11 was both capture and biotinylated detection antibody in a sandwich ELISA of trichloroacetic acid-pretreated serum samples. Sera of patients with 7-500 eggs/g of stool were positive by MAb 5H11-CCA sandwich ELISA. Stool egg counts and CCA serum levels correlated (r = .52), and CCA levels decreased by 4 weeks after praziquantel treatment in patients with pretreatment egg counts of greater than or equal to 50/g of stool (P less than .05). Sera of Schistosoma haematobium-infected patients, uninfected individuals, and most patients with other helminths were negative in this assay. The MAb 5H11-CCA sandwich ELISA appears sensitive and specific for immunodetection of active schistosomiasis mansoni and useful for monitoring its chemotherapy.

Schistosoma mansoni: detection of circulating antigens in murine schistosomiasis by antigen-capture sandwich ELISA using a monoclonal antibody.

A monoclonal antibody (MAb) 5H11/B1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mg/kg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5H11/B1 assay in light and heavy infection (r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the 5H11/B1 sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis using this assay are in progress.

Monoclonal antibody neutralization of unmanipulated Chlamydia trachomatis serovar A infection of human epithelioid cells (A-431).

A human epithelioid cell line (A-431) was tested in parallel with McCoy fibroblast cells for the growth of trachoma-related serovar A Chlamydia trachomatis without centrifugation or cycloheximide addition. A-431 cells were 4-7 times more susceptible to infection with serovar A than McCoy cells in such unmanipulated cultures. Murine monoclonal antibodies (MAbs) developed against serovar A were then evaluated for their ability to inhibit unmanipulated serovar A infectivity of A-431 cells. Two of seven MAbs tested neutralized infectivity by more than 50%. An IgG2a MAb (2C8) that is specific for serovar A, and another IgG2a MAb (4E3) that reacts equally with serovars A and L2 neutralized infectivity of serovar A by 72.2 +/- 3.7% and 56.0 +/- 5.8% (mean +/- SEM of 7 experiments) respectively. Mouse immune serum (MIS) raised against serovar A elementary bodies (EB) neutralized infectivity of serovar A by 76.0 +/- 4.9% (mean +/- SEM of 7 experiments). Immunoblot detection of serovar A EB polypeptides separated by SDS-PAGE indicated that 2C8 reacted with a 16 kD and 4E3 reacted with a 12 kD polypeptide while MIS reacted with several polypeptides including the major outer membrane protein (MOMP). These studies show that the human epithelioid cell line A-431 is a more susceptible host than McCoy cells in unmanipulated cultures, and that 2 MAbs neutralize serovar A infectivity of A-431 cells. Identification of antigenic moieties of importance in unmanipulated chlamydial infections may help in the development of potential vaccines against trachoma.

Immune responses of mice after conjunctival exposure to Chlamydia trachomatis serovar A.

CBA/J mice were inoculated in the lower conjunctival sac with live elementary bodies (EBs) of Chlamydia trachomatis serovar A. Recovery of chlamydia after exposure was done by culture of conjunctival swabs and draining lymph node (D-LN) cells in McCoy cells grown on coverslips in isolation vials. Cellular immune responsiveness was measured by lymphocyte proliferation assay of D-LN cells stimulated with irradiated EBs of serovars A, C, or L2. Humoral immunity was measured by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Chlamydia were consistently isolated from the conjunctiva and from the D-LN at 1 and 7 days after exposure respectively. Intermittent isolations were obtained from the conjunctiva up to day 4 and from the D-LN up to day 14 after a single exposure. Serovar A EB-stimulated lymphocyte proliferation was strong by 1 week after conjunctival exposure, but by 4 and 5 weeks, blastogenic responsiveness was very low. This lack of responsiveness may reflect a state of immunosuppression. Responses to serovars C and L2 EBs were consistently lower than to serovar A EBs. Serum IgG antichlamydia antibodies were not detected by ELISA until 2 weeks after exposure, peaked by 4-5 weeks, and decreased between 5 and 7 weeks after exposure. The IgM response was minimal at all times tested. There was, however, a modest increase in IgM antibodies at 3 and 5-7 weeks after exposure. Immunoblot analysis showed reactivity of mouse serum antibodies with polypeptide bands of 30, 41, and 52 kD at 3 and 4 weeks post exposure and predominantly with the 52 kD moiety at 5 weeks post exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of trachoma in school children and ophthalmological outpatients in rural Egypt.

The prevalence of trachoma in school children and ophthalmological patients in rural villages of the Qalyub Governorate of Egypt was determined by clinical and laboratory diagnostic procedures and reported as mild, moderate, or severe according to the WHO classification scheme. Of 777 primary school students examined in 3 villages, 204 (26%) had clinically active trachoma. The overall prevalence of the disease in this population ranged from 16% to 35%. The prevalence of infection was higher in younger groups and decreased throughout primary school. Of 312 patients with ocular complaints examined at the village outpatient clinics, 100 (32%) had trachoma infections. Monoclonal FA staining showed higher sensitivity in detecting positive cases of trachoma than did Giemsa staining. This study has shown that trachoma is still prevalent in rural Egypt and that the monoclonal FA staining is a relatively sensitive and practical test for the laboratory diagnosis of trachoma in a field study, where reasonable facilities for culture diagnosis are not available.

Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A.

Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization by human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.

Immune responses and immunoregulation in relation to human schistosomiasis in Egypt. III. Immunity and longitudinal studies of in vitro responsiveness after treatment.

Immune responsiveness of schistosomiasis patients was assayed longitudinally, before and for two years after chemotherapeutic treatment with praziquantel, by in vitro peripheral blood mononuclear cell (PBMN) proliferation upon exposure to phytohaemagglutinin (PHA), or soluble schistosomal antigenic preparations from eggs (SEA), adult worms (SWAP) or cercariae (CAP). Parallel faecal and urine examinations documented the infection status of the patients during this time. Treatment resulted in substantially increased responsiveness to the schistosome-derived materials but not to PHA or C. albicans extract. The responses to SEA, SWAP, and CAP often remained elevated for one to two years after treatment. However, those patients who became reinfected had significantly lower PBMN responses to SEA or CAP at the time of the last blastogenesis assay before the observation that they were again stool-positive for Schistosoma mansoni eggs. No other demonstrable differences (such as age, sex, household location, pre-treatment intensities of infection or occupation) were observed between those who remained uninfected for at least two years (resistant?) and those who became reinfected during this time (susceptible?).

Immune responses and immunoregulation in relation to human schistosomiasis in Egypt. II. Cimetidine reversal of histamine-mediated suppression of antigen-induced blastogenesis.

The effect of histamine on cell-mediated immune responses of chronic schistosomiasis patients was tested by peripheral blood mononuclear cell (PBMN) reactions to phytohemagglutinin-P (PHA) and soluble schistosomal antigenic preparations derived from eggs (SEA) or adult worms (SWAP). PBMN responses to PHA were suppressed by exogenous histamine (10(-5)M), and the addition of cimetidine (CIM) (10(-4)M), an H2-receptor antagonist, reversed this suppressive effect. Histamine primarily suppressed PBMN responses to suboptimal and optimal PHA concentrations. Exogenous histamine (10(-5)M) also suppressed PBMN responses of 27 schistosomiasis patients to SEA and SWAP, respectively. The addition of CIM (10(-4)M) to suppressed cultures reversed the effect of exogenous histamine. Most importantly, the addition of CIM to schistosomal antigen-induced cultures, without exogenous histamine, significantly increased patients' PBMN responses to SEA and SWAP. The mean optimal increase in SEA responses of 19 patients was 390%. With SWAP-induced responses of 21 patients this increase was 165%. The use of 10(-4)M diphenhydramine (DPH), an H1-receptor antagonist, resulted in general suppression of both PHA-induced and schistosomal antigen-induced PBMN responses. Lower concentrations of DPH lead to variable responses but did not result in consistent abrogation of the histamine-induced suppression. These data imply that an histamine-induced, H2-receptor-mediated suppressor circuit often helps modulate antigen-specific responsiveness of PBMN from patients with chronic schistosomiasis.

Evaluation of natural killer activity in human schistosomiasis.

Natural killer (NK) activity was assayed in peripheral blood mononuclear cells of patients with schistosomiasis, of patients following treatment, and of uninfected control subjects. The patient populations were from villages in the Qalyub Province, Egypt and around Belo Horizonte , Brazil. NK activity was assayed by the cytotoxicity of 51Cr-labelled K562 target tumor cells. In neither infected population were significant alterations from normal levels found in the percent cytotoxicity per 10(6) cells, or in the lytic units that expressed 25% cytotoxicity. Likewise, prior treatment (2 and 6 months previously) did not alter the group NK activity detected. Similarly, in the Egyptian study there was no difference in the percentage of large granular lymphocytes between the infected and uninfected groups. In parallel studies in Egyptian and Brazilian schistosomiasis patients we did not find any evidence that this chronic infection consistently altered circulating NK activity.

The effects of indomethacin on in vitro peripheral blood mononuclear cell reactivity in human schistosomiasis.

The peripheral blood mononuclear cell (PBMN) proliferative responses of cells from patients with schistosomiasis were studied in the presence and absence of indomethacin in the culture medium. PBMN cultures were exposed to antigenic extracts of either adult S. mansoni worms (SWAP) or cercariae (CAP), and assayed for the incorporation of tritiated thymidine. More than 70% of the 48 patients studied with SWAP and the 40 patients studied with CAP, were not substantially effected by the addition of indomethacin to the cultures. The remainder (less than 30%) was augmented more than 50% by indomethacin and comprise a group which gave initially low responses to these antigenic preparations. Further analysis indicated that in some schistosomal patients the effect of an adherent suppressor cell population may, in part, be based on a prostaglandin-mediated, indomethacin-sensitive suppressive mechanism. However, the majority of patients, most of whom display adherent suppressor cells, are unaffected by indomethacin. Apparently, other adherent cell suppressor mechanisms are responsible for the regulation observed.

Immune responses and immunoregulation in relation to human schistosomiasis in Egypt. I. Effect of treatment on in vitro cellular responsiveness.

Cell mediated immune reactivity of chronic schistosomiasis patients was tested in vitro by peripheral blood mononuclear cell (PBMN) responses against phytohemagglutinin P (PHA), Candida albicans extract, soluble schistosomal antigenic preparations derived from eggs (SEA), adult worms (SWAP) and cercariae (CAP), before and after treatment of the patients with parziquantel. The patient population was from villages in the Qalyub province, Egypt, that are endemic for Schistosoma mansoni and S. haematobium. Patients were studied immediately before, and at 1, 3, 6, and 9 months after chemotherapy. Egg counts were done on stool and urine specimens taken simultaneously with blood samples. There was a significant increase in PBMN responses to SWAP and CAP but not to SEA, PHA or C. albicans in 27 patients (age 8-65) 1 month after treatment. Eleven patients treated 1.5 years previously did not show such elevated responses 1 month after re-treatment. Three months after treatment higher mean responses were observed to SWAP, CAP, SEA, and PHA, but not to C. albicans in 24 patients (age 6-26). Significant increases in PBMN responses to SWAP and CAP, but not to SEA, PHA or C. albicans were obtained at 6 months after treatment in 12 patients (age 6-30). By 9 months after treatment in a group of 11 patients (age 8-25) SWAP and CAP responses were still elevated as were SEA and C. albicans induced reactivities. The PBMN responses of 10 patients were followed longitudinally at pretreatment, 3-, 6-, and 9-month post-treatment times. In general, elevated responses were maintained throughout this period to the schistosomal preparations. Unrelated responses occasionally fluctuated but were not consistently altered over time.

Modulation of mitogen-induced proliferation of autologous peripheral blood lymphocytes by human alveolar macrophages.

Experiments were carried out to determine the effect of cocultivation of T-cell-enriched human peripheral blood lymphocytes with autologous alveolar macrophages on mitogen-induced proliferation as determined by [(3)H]thymidine uptake. Cells obtained by fiberoptic bronchoscopy and saline bronchial lavage from 14 normal volunteers were enriched for macrophages by adherence in plastic dishes for 1 h in RPMI 1640 medium supplemented with 10% fetal calf serum. Nonadherent mononuclear cells were prepared from heparinized venous blood after Ficoll-Hypaque sedimentation by passage over nylon wool columns. T-cell-enriched populations were incubated with and without alveolar macrophages, either in the presence or absence of phytohemagglutinin. In these experiments, the number of lymphocytes was held constant (10(5) per well), while the number of alveolar macrophages was varied (0.1 x 10(5) to 4.0 x 10(5) per well). Alveolar macrophages generally tended to stimulate phytohemagglutinin-induced lymphoproliferation at lymphocyte/macrophage ratios of 10:1 but consistently and significantly suppressed proliferation at ratios which approach those usually observed in recovered human bronchial lavage fluid, namely, 1:4. The suppressive effect of alveolar macrophages was observed as early as 48 h after culture initiation, while the magnitude of suppression increased with time. Suppression did not appear to be due to alteration in lymphocyte viability, nor was it sensitive to indomethacin. These results indicate that human alveolar macrophages can modulate the in vitro proliferative response of autologous peripheral blood lymphocytes. This observation may have relevance to interactions between alveolar macrophages and bronchial lymphocytes in the human lung in vivo.

Human alveolar macrophages: effects of endotoxin in vitro.

Experiments were performed to evaluate the in vitro effects of Escherichia coli lipopolysaccharide on viability and function of human alveolar macrophages. Alveolar macrophages were obtained by fiberoptic bronchoscopy and saline bronchial lavage from 12 normal, nonsmoking volunteers. Cells were incubated with different concentrations of E. coli endotoxin for 1 and 24 h. Endotoxin (10 microgram/ml and more) was cytotoxic for alveolar macrophages after 24 h of incubation and induced significant inhibition of phagocytosis, adherence, and spreading. The effects of endotoxin on alveolar macrophage viability and function were dose and time dependent and were not influenced by indomethacin. Thus, human alveolar macrophages, like other mononuclear phagocytes, are extremely sensitive to endotoxin effects; these observations may be relevant in conditions in which endotoxin may be in contact with alveolar macrophages in vivo: endobronchial infections with gram-negative organisms, byssinosis, chronic bronchitis of grain handles, and humidifier fever.

Equine leptospirosis with some clinical observations.

In a serologic survey on equine leptospirosis in Egypt, the following incidences of leptospiral serosensitivity were found: 1. Hospitalised horses 65/113 (57.5 %). 2. Hospitalised donkeys 90/125 (72.0 %). 3. Apparently healthy horses 21/72 (29.1 %). Sera of these animals were mostly reacting to serotypes butembo, pomona, icterohemorragiae, and grippotyphosa. Equine in Egypt are close animals to humans and may constitute a potential source of leptospiral infection. From the clinical point of view, it is very possible that ocular, hoof lesions and icterus in equines would be expected with leptospiral titres.