Immunization of rabbits with recombinant Treponema pallidum surface antigen 4D alters the course of experimental syphilis

Rabbits immunized with the protease resistant recombinant Treponema pallidum surface-associated Ag 4D showed an altered course of experimental syphilis after intradermal challenge with virulent T. pallidum. Vaccination trials using three different protocols were examined. In one experiment, a combined i.m.-i.v. immunization protocol was compared with an i.m. injection schedule. Four of five rabbits immunized by the i.m.-i.v. protocol developed morphologically atypical lesions at each of 16 sites, whereas the i.m. injected animals showed no evidence of attenuated disease. Moreover, immunization by both protocols elicited equally high ELISA titers of 4D-specific IgG antibody suggesting that cellular immune mechanisms may have been involved. In an effort to augment cell-mediated responses to 4D, seven rabbits received i.v. immunizations with an emulsion of 4D, purified BCG cell wall skeletons, and trehalose dimycolate in an oil-microdroplet form (4D-cell wall skeleton-trehalose dimycolate). Splenic lymphocytes from three representative immunized animals had a strong in vitro proliferative response to 4D as compared to controls. Further, all animals developed high anti-4D titers in response to immunization. After challenge with virulent T. pallidum, six of the seven rabbits developed an early cutaneous response (3-8 days) at the sites of inoculation consistent with a hypersensitivity reaction followed by morphologically atypical lesions. Aspirates from each of three representative atypical lesions were devoid of treponemes by darkfield examination at day 14, whereas motile T. pallidum were observed in aspirates from four of five control sites. Between days 20 and 24, lesions in the 4D-cell wall skeleton-trehalose dimycolate immunized rabbits began to enlarge and become more typical in appearance; aspirates from six of seven representative lesions were darkfield positive. We conclude that 1) immunization with the r4D Ag alters the course of experimental syphilis in a manner consistent with previously defined parameters of partial protection and 2) immunizations protocols containing an i.v. component are more effective than protocols using the i.m. route alone.     

Are selective serotonin reuptake inhibitors cardiac teratogens? Echocardiographic screening of newborns with persistent heart murmur

BACKGROUND : Selective serotonin reuptake inhibitors (SSRIs) have been suspected of cardiac teratogenicity, but reports have been inconsistent. Our aim was to investigate the rate of nonsyndromic congenital heart defects in newborns exposed in utero to SSRIs compared with unexposed controls. METHODS : This prospective study of women who gave birth at our tertiary center from 2000 to 2007 yielded 235 women who reported first‐trimester SSRI use during pregnancy. All newborns born during the study period and found to have a persistent cardiac murmur on day 2 or 3 of life were referred for examination by a pediatric cardiologist and by echocardiography. The findings were compared between the newborns who were exposed to SSRIs and those who were not. RESULTS : Nonsyndromic congenital heart defects were identified by echocardiography in 8 of 235 (3.40%) newborns exposed in utero to SSRIs and in 1083 of 67,636 (1.60%) non‐exposed newborns. The difference in prevalence between the two groups was significant (relative risk, 2.17; 95% confidence interval, 1.07–4.39). The prevalence rates for paroxetine and fluoxetine exposure were 4.3% and 3.0%, respectively. All cardiac defects in the study group were mild: ventricular septal defect (6), bicuspid aortic valve (1) and right superior vena cava to coronary sinus (1). CONCLUSIONS : Newborns exposed in utero to SSRIs, have a twofold higher risk of mild nonsyndromic heart defects than unexposed infants. The data suggest that women who require SSRI treatment during pregnancy can be reassured that the fetal risk is low and possible cardiac malformations will probably be mild. Late‐targeted ultrasound and fetal echocardiography at 22 to 23 weeks' gestation are recommended in this patient group. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.     

Detection and functional characterization of early appearing antibodies in rabbits with experimental syphilis

Following testicular infection of rabbits with Treponema pallidum, different antibodies become detectable initially at the time of healing. Experiments were performed to determine a functional role for these antibodies. Rabbits were sacrificed after 4-8 days. Treponemal numbers steadily increased for 10-12 days. Thereafter, host defenses were sufficiently stimulated to begin clearing the organisms. Antibodies in serum and antibodies localized at the site of infection were quantitated using radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) techniques. Anti-treponemal IgG was detected as early as day 4. Quantities of antibody correspondingly increased with time following infection. Treponema pallidum was harvested 7 and 14 days postinfection and tested for surface antibodies. With increasing days postinfection, more antibody was found on the organisms. Two functional properties of these antibodies were shown. Sera from 24 of 45 rabbits infected for 14 days immobilized T. pallidum in the presence of complement and 14-day sera blocked the attachment of T. pallidum to tissue culture cells. We suggest that antibody-mediated, complement-dependent immobilization of T. pallidum and blockage of attachment are at least partially responsible for healing of testicular lesions.     

Impact of BVDV infection of white-tailed deer during second and third trimesters of pregnancy

While it has been demonstrated that persistent bovine viral diarrhea virus (BVDV) infections can be established in white-tailed deer (Odocoileus virginianus) following in utero exposure in the first trimester of gestation, there is little to no information regarding the outcome of infection in later stages of pregnancy in deer. Our goal was to observe the impact of infection of white-tailed deer in the second and third trimesters of pregnancy. Five white-tailed deer in the second trimester of pregnancy and four in the third trimester were infected with a BVDV type 2 virus previously isolated from a BVDV-infected deer harvested from the wild. Infection of deer in the second trimester of pregnancy resulted in loss of the pregnancy in three of five deer. Fawns born to the two remaining deer appeared normal and were born BVDV antigen-negative with neutralizing serum antibodies against BVDV. Infection of does in the third trimester of pregnancy did not result in fetal death or persistent infection and all does gave birth to live, healthy fawns that were BVDV antigen-negative and born with antibodies against BVDV. These results, combined with those previously reported regarding BVDV infection in the first trimester of pregnancy, suggest that the impact of BVDV infection of pregnant white-tailed deer is very similar to that observed in pregnant cattle.     

Socio-demographic characteristics and lifestyle habits of pregnant women smokers

The aim of this study was to describe anthropometric, clinical, socio-demographic characteristics and lifestyle habits of pregnant smokers in comparison to pregnant nonsmokers. During years 1999-2003, 1,435 pregnant smokers and 4,772 pregnant nonsmokers were interviewed after delivery with a questionnaire. They were recorded clinical, anthropometric and socio-demographic data, smoking status, labor outcome, maternal and fetal hemoglobin concentrations for each patient. The two groups were comparable in anthropometric and clinical characteristics, duration of pregnancy and mode of delivery, except for birth weights, which were significantly lower in newborns of smokers. Maternal hemoglobin concentrations were significantly lower in smokers, but fetal hemoglobin concentrations were significantly higher in babies of smokers. The proportion of pregnant women who smoked during pregnancy was higher among urban women, among women with lower educational level and among unemployed subjects in comparison with nonsmokers. The pregnant women who smoked during pregnancy were more often caffeine and alcohol consumers. To further reduce smoking during pregnancy it is important to continue to promote smoking cessation among teenagers.     

先天性巨细胞病毒感染的研究进展

巨细胞病毒(cytomegalovirus, CMV)在世界范围内均有较高的感染率,是最常见的先天性感染。先天性CMV感染可继发于母体原发性感染或非原发性感染。高达40%～50%的感染新生儿是在妊娠早期原发感染, 之后出现长期后遗症,主要包括先天性CMV感染相关听力损伤和神经后遗症。血清学检查对于确定原发性CMV感染至关重要。产前超声检查发现胎儿异常要警惕先天性CMV感染的可能性,磁共振成像有助于发现CMV相关脑异常。羊膜穿刺术是诊断胎儿CMV感染的金标准。加强育龄妇女及孕妇的卫生健康知识教育、减少CMV感染及抗病毒治疗是目前预防先天性CMV感染的主要措施。更昔洛韦及缬更昔洛韦是目前治疗CMV感染最有效的药物。高免疫球蛋白及CMV疫苗预防先天性 CMV 感染的作用尚无明确结论。     

Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: Relationship to the development of opsonizing antibodies

Abstract Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum . These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion ( P < 0.001) and macrophage-mediated killing ( P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.     

Congenital Chagas disease: an update

Congenital infection with Trypanosoma cruzi is a global problem, occurring on average in 5% of children born from chronically infected mothers in endemic areas, with variations depending on the region. This presentation aims to focus on and update epidemiological data, research methods, involved factors, control strategy and possible prevention of congenital infection with T. cruzi. Considering that etiological treatment of the child is always effective if performed before one year of age, the diagnosis of infection in pregnant women and their newborns has to become the standard of care and integrated into the surveillance programs of syphilis and human immunodeficiency virus. In addition to the standard tests, polymerase chain reaction performed on blood of neonates of infected mothers one month after birth might improve the diagnosis of congenital infection. Recent data bring out that its transmission can be prevented through treatment of infected women before they become pregnant. The role of parasite genotypes and host genetic factors in parasite transmission and development of infection in foetuses/neonates has to be more investigated in order to better estimate the risk factors and impact on health of congenital infection with T. cruzi.     

CONGENITAL SYPHILIS IN CALIFORNIA

Routine serologic tests for syphilis (as required by California law governing prenatal examination) and penicillin therapy during pregnancy for infected mothers have been major factors in the prevention of congenital syphilis in California during the past ten years. In 1940 one of each 822 infants had the disease, as indicated by morbidity reports of congenital syphilis in infants under the age of one year. In 1950 the ratio was one in 8,148. To determine why congenital syphilis continues to occur, a study of the 134 cases reported over a two-year period was made with the cooperation of local health officers and practicing physicians. It showed that in 76 per cent of cases the mother did not consult a physician prior to delivery or reported so late in pregnancy that the infant was born before adequate penicillin therapy could be given. In another 15 per cent syphilis developed in the mother during pregnancy after a negative reaction to a prenatal serologic test. The other 9 per cent of cases were due to various factors, such as infectious relapse or reinfection in previously adequately treated mothers. The study indicated that most cases occur in the lower socioeconomic population groups. Seventy-four per cent of cases were in infants delivered in county hospitals.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.     

3670 例早期妊娠妇女梅毒血清学检查及处理

目的:了解早期妊娠妇女梅毒感染情况,采取防治措施,减少先天梅毒的发生。方法:采用梅毒明胶颗粒试验(TPPA)和梅毒甲苯胺红试验(TRUST)对早期妊娠妇女进行梅毒血清学检查,两项结果均阳性者诊为妊娠期梅毒,可采取驱梅治疗或终止妊娠。结果:3670例早期妊娠妇女中,确诊为妊娠期梅毒的29例,其中TRUST滴度大于1:8的有8例,小于1:8的有21例;有12人接受驱梅治疗,有17人选择终止妊娠。结论:在早期妊娠妇女中进行梅毒血清学检查,可及早采取措施,减少先天梅毒的发生。     

Congenital transmission of Schistosoma japonicum in the rabbit

Fourteen pregnant rabbits were each infected with 300 cercariae of Schistosoma japonicum and divided into two groups. Group M (n = 8) was infected during mid-gestation (the organogenetic stage) and group L (n = 6) was infected during late-gestation (the post-organogenetic stage). Mother rabbits and rabbit kittens were killed 45-60 days after infection and perfused in order to obtain worm counts. Furthermore, faecal egg counts and tissue egg counts from livers were obtained from the mother rabbits as well as the rabbit kittens. All mother rabbits became infected harbouring 207.6 +/- 20.2 and 220.0 +/- 27.5 adult worms in group M and L, respectively. In groups M and L, 13.5% and 46.7% of the kittens were infected, respectively. In 12 of 14 litters at least one kitten was infected. The infected kittens harboured between one and three adult S. japonicum. The livers of the kittens infected with a worm pair displaced lesions as a result of egg deposition. The results, therefore, show that congenital transmission of S. japonicumcan occur in rabbits. The close anatomical resemblance between the rabbit and human placenta may be indicative of the presence of congenital transmission of S. japonicum infection in humans.     

Development of cellular immunity to individual soluble antigens of Treponema pallidum during experimental syphilis

The contribution of individual specific molecules of Treponema pallidum subspecies pallidum to cellular immunity in experimental syphilis was evaluated by combining the techniques of Ag identification and purification with the lymphocyte proliferation assay. Proliferative responses of splenic lymphocytes from syphilitic rabbits to complex treponemal Ag and Con A were vigorous throughout the course of intratesticular infection (6, 10, 17, 30, and 210 days). Normal rabbits did not respond to any treponemal preparations and all rabbits failed to respond to normal rabbit testicular Ag (NRT). Seven defined treponemal Ag (47 kDa, 37 kDa, 35, 33-kDa, 30-kDa, 14 kDa, and 12 kDa) stimulated lymphocytes from infected rabbits. Cellular responses to the 37-kDa and 30-kDa fractions were evident by day 6 of infection and responses to the 35, 33-kDa and 14-kDa Ag were first detected on day 10; responsiveness to these Ag continued throughout the observation period. Cellular responses to the 47-kDa molecule were detectable but lower when compared with other individual Ag. Responsiveness to the 12-kDa Ag was not evident until 7 mo postinfection. Specific immunoblot reactivity of serum from rabbits used in this study generally correlated with the development of cellular reactivity to individual Ag of T. pallidum.     

The embryotoxicity of gossypol

In utero development was analyzed in pregnancies that resulted from matings between gossypol-treated male rats and untreated female rats and in pregnancies in which gossypol was administered to the pregnant rat only. Gossypol treatment of males had no effect on the outcome of pregnancy. There was no significant effect on resorption, fetal growth, or malformation rate. Similarly, gossypol administered to pregnant dams at stages during organogenesis had no observable effect on pregnancy. Under the conditions of this experiment, gossypol administered to either the breeding male rat or the pregnant female rat had no demonstrable adverse effect on development in utero.     

Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.     

Experimental congenital disease with simian cytomegalovirus in rhesus monkeys

Cytomegalovirus (CMV) infections occur worldwide and are responsible for severe damage to the child in from one to five newborns per 20,000 births. Animal models of congenital CMV infection resulting in disease have been developed in mice and guinea pigs. We report here the development of ventricular dilatation and leptomeningitis in rhesus monkeys, Macaca mulatta, following intrauterine infection with rhesus cytomegalovirus (RCMV). Central nervous system (CNS) lesions were associated with low cytomegalovirus fluorescent antibody titers in affected fetuses. In several infected animals, RCMV was isolated at necropsy from neural and nonneural tissues taken shortly after birth. This model allows investigators to study the pathogenesis and prevention of CNS changes following RCMV infection.     

Global Estimates of Syphilis in Pregnancy and Associated Adverse Outcomes: Analysis of Multinational Antenatal Surveillance Data

Background

The World Health Organization initiative to eliminate mother-to-child transmission of syphilis aims for ≥90% of pregnant women to be tested for syphilis and ≥90% to receive treatment by 2015. We calculated global and regional estimates of syphilis in pregnancy and associated adverse outcomes for 2008, as well as antenatal care (ANC) coverage for women with syphilis.

Methods and Findings

Estimates were based upon a health service delivery model. National syphilis seropositivity data from 97 of 193 countries and ANC coverage from 147 countries were obtained from World Health Organization databases. Proportions of adverse outcomes and effectiveness of screening and treatment were from published literature. Regional estimates of ANC syphilis testing and treatment were examined through sensitivity analysis. In 2008, approximately 1.36 million (range: 1.16 to 1.56 million) pregnant women globally were estimated to have probable active syphilis; of these, 80% had attended ANC. Globally, 520,905 (best case: 425,847; worst case: 615,963) adverse outcomes were estimated to be caused by maternal syphilis, including approximately 212,327 (174,938; 249,716) stillbirths (>28 wk) or early fetal deaths (22 to 28 wk), 91,764 (76,141; 107,397) neonatal deaths, 65,267 (56,929; 73,605) preterm or low birth weight infants, and 151,547 (117,848; 185,245) infected newborns. Approximately 66% of adverse outcomes occurred in ANC attendees who were not tested or were not treated for syphilis. In 2008, based on the middle case scenario, clinical services likely averted 26% of all adverse outcomes. Limitations include missing syphilis seropositivity data for many countries in Europe, the Mediterranean, and North America, and use of estimates for the proportion of syphilis that was “probable active,” and for testing and treatment coverage.

Conclusions

Syphilis continues to affect large numbers of pregnant women, causing substantial perinatal morbidity and mortality that could be prevented by early testing and treatment. In this analysis, most adverse outcomes occurred among women who attended ANC but were not tested or treated for syphilis, highlighting the need to improve the quality of ANC as well as ANC coverage. In addition, improved ANC data on syphilis testing coverage, positivity, and treatment are needed. Please see later in the article for the Editors'' Summary