Changes in Toxoplasma diagnosis.

The serological laboratory workload in detecting toxoplasma infection may be expected to change with changes in the clinical profile of patient populations. We have examined the clinical information and laboratory results for patients referred to the Scottish Toxoplasma Reference Laboratory in April-March 1999-2000 and 2009-2010. Numbers of patient sera submitted for testing were similar (1624 and 1552) but there was a change in the clinical profile, with a significant fall in patients with symptoms of current infection (612 versus 335; P<0.0001) and a significant rise in immunocompromised patients (275 versus 531; P<0.0001). Although the percentage of patient samples with toxoplasma antibody decreased (53.9% versus 37.5%; P<0.0001), the number of positives increased with age, demonstrating the continuing risk of toxoplasma infection. More cases of current infection were identified in 2009-2010 than in 1999-2000 (48 versus 35). This increase was significant both for all females with current infection (P=0.0253) and also for those in the childbearing 20-39 years age group (P=0.0388). Our literature search did not find any published information on toxoplasma workload in the last decade. In summary, we have shown that there have been significant changes in the laboratory diagnosis of toxoplasma infection but it is as important as ever that effective diagnostic strategies are maintained.

Antibody treatment promotes compensation for human cytomegalovirus-induced pathogenesis and a hypoxia-like condition in placentas with congenital infection.

Human cytomegalovirus (HCMV) is the major viral cause of birth defects worldwide. Affected infants can have temporary symptoms that resolve soon after birth, such as growth restriction, and permanent disabilities, including neurological impairment. Passive immunization of pregnant women with primary HCMV infection is a promising treatment to prevent congenital disease. To understand the effects of sustained viral replication on the placenta and passive transfer of protective antibodies, we performed immunohistological analysis of placental specimens from women with untreated congenital infection, HCMV-specific hyperimmune globulin treatment, and uninfected controls. In untreated infection, viral replication proteins were found in trophoblasts and endothelial cells of chorionic villi and uterine arteries. Associated damage included extensive fibrinoid deposits, fibrosis, avascular villi, and edema, which could impair placental functions. Vascular endothelial growth factor and its receptor fms-like tyrosine kinase 1 (Flt1) were up-regulated, and amniotic fluid contained elevated levels of soluble Flt1 (sFlt1), an antiangiogenic protein, relative to placental growth factor. With hyperimmune globulin treatment, placentas appeared uninfected, vascular endothelial growth factor and Flt1 expression was reduced, and sFlt1 levels in amniotic fluid were lower. An increase in the number of chorionic villi and blood vessels over that in controls suggested compensatory development for a hypoxia-like condition. Taken together the results indicate that antibody treatment can suppress HCMV replication and prevent placental dysfunction, thus improving fetal outcome.

More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

AIMS: To identify further Western blot bands that may be specific in the diagnosis of Lyme borreliosis. METHODS: The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined. RESULTS: 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test). CONCLUSION: Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

Toxoplasma tachyzoites from cell culture are more appropriate in some situations.

BACKGROUND: Laboratories traditionally culture toxoplasma tachyzoites in animals for testing and experimental use. This article considers why available cell culture methods are not used more often. AIM: To compare HeLa cell culture and animal culture for production of toxoplasma tachyzoites. METHODS: In 2000 HeLa culture replaced animal culture for continuous production of toxoplasma tachyzoites in the Scottish Toxoplasma Reference Laboratory. The performance of animal culture (1994-1998) was compared with HeLa culture (2004-2008). A PubMed search was carried out for 1998 and 2008 to assess the culture methods used in laboratories. RESULTS: Animal culture was able to produce higher yields of tachyzoites (10(9) from a cotton rat peritoneal harvest compared to 10(7) from a 75 cm(2) cell culture flask) but significantly more HeLa cultures were successful (93% versus 84%; p=0.025). There was no difference in the quality of tachyzoites from animal and HeLa cultures as demonstrated by the high levels of success in the dye test. HeLa culture offered significant advantages in flexibility and control. A review of the literature showed no significant change in the method of culture used in laboratories between 1998 and 2008 (p=0.36). CONCLUSION: The availability of cell culture methods and the increasingly stringent regulations on the use of animals have not resulted in a decline in the use of animal culture. Animals are necessary for certain experiments but many studies could use cell-culture-derived parasites.

Gene expression profiling of the human maternal-fetal interface reveals dramatic changes between midgestation and term.

Human placentation entails the remarkable integration of fetal and maternal cells into a single functional unit. In the basal plate region (the maternal-fetal interface) of the placenta, fetal cytotrophoblasts from the placenta invade the uterus and remodel the resident vasculature and avoid maternal immune rejection. Knowing the molecular bases for these unique cell-cell interactions is important for understanding how this specialized region functions during normal pregnancy with implications for tumor biology and transplantation immunology. Therefore, we undertook a global analysis of the gene expression profiles at the maternal-fetal interface. Basal plate biopsy specimens were obtained from 36 placentas (14-40 wk) at the conclusion of normal pregnancies. RNA was isolated, processed, and hybridized to HG-U133A&B Affymetrix GeneChips. Surprisingly, there was little change in gene expression during the 14- to 24-wk interval. In contrast, 418 genes were differentially expressed at term (37-40 wk) as compared with midgestation (14-24 wk). Subsequent analyses using quantitative PCR and immunolocalization approaches validated a portion of these results. Many of the differentially expressed genes are known in other contexts to be involved in differentiation, motility, transcription, immunity, angiogenesis, extracellular matrix dissolution, or lipid metabolism. One sixth were nonannotated or encoded hypothetical proteins. Modeling based on structural homology revealed potential functions for 31 of these proteins. These data provide a reference set for understanding the molecular components of the dialogue taking place between maternal and fetal cells in the basal plate as well as for future comparisons of alterations in this region that occur in obstetric complications.

Maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal Fc receptor-mediated transcytosis.

How human cytomegalovirus (CMV) reaches the fetus across the placenta is unknown. The major viral cause of congenital disease, CMV infects the uterine-placental interface with varied outcomes depending on the strength of maternal humoral immunity and gestational age. Covering the surface of villi that float in blood, syncytiotrophoblasts express the neonatal Fc receptor (FcRn) that transports IgG for passive immunity. Immunohistochemical analysis of early-gestation biopsy specimens showed an unusual pattern of CMV replication proteins in underlying villus cytotrophoblasts, whereas syncytiotrophoblasts were spared. Found in placentas with low to moderate CMV-neutralizing antibody titers, this pattern suggested virion transcytosis across the surface. In contrast, syncytiotrophoblasts from placentas with high neutralizing titers contained viral DNA and caveolin-1-positive vesicles in which IgG and CMV glycoprotein B co-localized. In villus explants, IgG-virion transcytosis and macrophage uptake were blocked with trypsin-treatment and soluble protein A. Quantitative analysis in polarized epithelial cells showed that FcRn-mediated transcytosis was blocked by the Fc fragment of IgG, but not F(ab')(2). Our results suggest that CMV virions could disseminate to the placenta by co-opting the receptor-mediated transport pathway for IgG. These findings could explain the efficacy of hyperimmune IgG for treatment of primary CMV infection during gestation and support vaccination.

Patterns of human cytomegalovirus infection in term placentas: a preliminary analysis.

BACKGROUND: Primary maternal CMV infection is the major risk factor for symptomatic congenital infection as maternal immunity reduces the risk of transmission to the fetus. Analysis of first trimester placentas showed that virus replicates in the uterus and is transmitted to the placenta causing focal infection. OBJECTIVES AND STUDY DESIGN: We examined 78 term placentas from uncomplicated deliveries for the presence of CMV DNA and evaluated evidence of infection by means of immunohistological and serological analysis. RESULTS: PCR analysis of villus biopsy samples and decidua showed that CMV DNA was present in 62% of tissues. Seven placentas with neutralizing titers were further examined by immunohistology for expression of viral proteins. In placentas with high levels of CMV DNA, fetal blood vessels in the villus core contained neutrophils with viral replication proteins, and macrophages/dendritic cells with glycoprotein B (gB). Cord blood samples from 1 of 11 placentas contained CMV DNA, an indication of replication in the fetal compartment. In placentas with low levels of viral DNA, macrophage/dendritic cells in the villus core contained CMV gB. This pattern was comparable to that seen in early gestation placentas from women with strong neutralizing antibodies. CONCLUSIONS: The results show CMV replication proteins in focal areas of the placenta, implying virus transmission to the fetal circulation. These preliminary results suggest that the incidence of asymptomatic congenital CMV infection might be higher than currently estimated.

Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders.

OBJECTIVE: To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ. DESIGN: We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs. RESULT(S): Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco's modified Eagle's medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype, were grown for more than 25 passages, expressed markers of stemness including Oct-3/4, Tra 1-60, Tra 1-80, and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to growth on a human placental laminin substrate in a defined medium. CONCLUSION(S): To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders.

Insights into viral transmission at the uterine-placental interface.

During human gestation, viruses can cause intrauterine infections associated with pregnancy complications and fetal abnormalities. The ability of viruses to spread from the infected mother to the fetus arises from the architecture of the placenta, which anchors the fetus to the uterus. Placental cytotrophoblasts differentiate, assume an endothelial phenotype, breach uterine blood vessels and form a hybrid vasculature that amplifies the maternal blood supply for fetal development. Human cytomegalovirus - the major cause of congenital disease - infects the uterine wall and the adjacent placenta, suggesting adaptation for pathogen survival in this microenvironment. Infection of villus explants and differentiating and/or invading cytotrophoblasts offers an in vitro model for studying viruses associated with prenatal infections.

Viral and bacterial pathogens at the maternal-fetal interface.

We studied the incidence of pathogenic bacteria and concurrent infections with human cytomegalovirus (CMV) and herpes simplex virus (HSV) type 1 and 2 in biopsy samples from the placenta and decidua of women with healthy pregnancies. By polymerase chain reaction analysis, we found that 38% of placental samples were positive for selected bacteria and viruses. CMV, HSV-1, and HSV-2 were detected in isolation or with bacteria in first- and second-trimester samples. Certain bacteria were detected more often during the second trimester than during the first--Ureaplasma urealyticum, Mycoplasma hominis, and Gardnerella/Bifidobacterium species. In paired samples from first-trimester tissues, the detection rate for viruses, compared with most bacteria, was higher in the decidua than in the adjacent placenta. In contrast, bacteria were more frequently detected in placenta. Analyses of immunoglobulin G isolated from the placenta support the hypothesis that immune responses suppress CMV reactivation in the presence of pathogenic bacteria at the maternal-fetal interface.

Human cytomegalovirus interleukin-10 downregulates metalloproteinase activity and impairs endothelial cell migration and placental cytotrophoblast invasiveness in vitro.

At the uterine-placental interface, fetal cytotrophoblasts invade the decidua, breach maternal blood vessels, and form heterotypic contacts with uterine microvascular endothelial cells. In early gestation, differentiating- invading cytotrophoblasts produce high levels of matrix metalloproteinase 9 (MMP-9), which degrades the extracellular matrix and increases the invasion depth. By midgestation, when invasion is complete, MMP levels are reduced. Cytotrophoblasts also produce human interleukin-10 (hIL-10), a pleiotropic cytokine that modulates immune responses, helping to protect the fetal hemiallograft from rejection. Human cytomegalovirus (CMV) is often detected at the uterine-placental interface. CMV infection impairs cytotrophoblast differentiation and invasion, altering the expression of the cell adhesion and immune molecules. Here we report that infection with a clinical CMV strain, VR1814, but not a laboratory strain, AD169, downregulates MMP activity in uterine microvascular endothelial cells and differentiating-invading cytotrophoblasts. Infected cytotrophoblasts expressed CMV IL-10 (cmvIL-10) mRNA and secreted the viral cytokine, which upregulated hIL-10. Functional analyses showed that cmvIL-10 treatment impaired migration in endothelial cell wounding assays and cytotrophoblast invasion of Matrigel in vitro. Comparable changes occurred in cells that were exposed to recombinant hIL-10 or cmvIL-10. Our results show that cmvIL-10 decreases MMP activity and dysregulates the cell-cell and/or cell-matrix interactions of infected cytotrophoblasts and endothelial cells. Reduced MMP activity early in placental development could impair cytotrophoblast remodeling of the uterine vasculature and eventually restrict fetal growth in affected pregnancies.

Human cytomegalovirus transmission from the uterus to the placenta correlates with the presence of pathogenic bacteria and maternal immunity.

Prenatal cytomegalovirus infection may cause pregnancy complications such as intrauterine growth restriction and birth defects. How virus from the mother traverses the placenta is unknown. PCR analysis of biopsy specimens of the maternal-fetal interface revealed that DNA sequences from cytomegalovirus were commonly found with those of herpes simplex viruses and pathogenic bacteria. Cytomegalovirus DNA and infected cell proteins were found more often in the decidua than in the placenta, suggesting that the uterus functions as a reservoir for infection. In women with low neutralizing titers, cytomegalovirus replicated in diverse decidual cells and placental trophoblasts and capillaries. In women with intermediate to high neutralizing titers, decidual infection was suppressed and the placenta was spared. Overall, cytomegalovirus virions and maternal immunoglobulin G were detected in syncytiotrophoblasts, villus core macrophages, and dendritic cells. These results suggest that the outcome of cytomegalovirus infection depends on the presence of other pathogens and coordinated immune responses to viral replication at the maternal-fetal interface.

Focal adhesion kinase is required for blood vessel morphogenesis.

The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is a point of convergence for signals from extracellular matrix, soluble factors, and mechanical stimuli. Targeted disruption of the fak gene in mice leads to death at embryonic day 8.5 (E8.5). FAK-/- embryos have severely impaired blood vessel development. Gene expression and in vitro differentiation studies revealed that endothelial cell differentiation was comparable in FAK-/- and wild-type E8.5 embryos. We examined the role of FAK in blood vessel morphogenesis using an in vitro tubulogenesis assay and three different culture systems: FAK+/+ and FAK-/- embryoid bodies, FAK+/+ and FAK-/- endothelial cells, and human umbilical vein endothelial cells expressing antisense FAK, a dominant-negative fragment of FAK, or wild-type FAK. In all of these systems, endothelial cells deficient in FAK expression or function displayed a severely reduced ability to form tubules in Matrigel. These studies demonstrate clearly that the vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular networks, rather than from defects in tissue-specific differentiation.

Association of antiretroviral resistance genotypes with response to therapy--comparison of three models.

Genotype-based resistance assays are commonly used to aid treatment in HIV-infected individuals failing antiretroviral therapy. The relationship between genotype and antiretroviral therapy comes mostly from in vitro assays of the response to a single drugs although there is a need for a prediction of clinical response to combination therapy. We have compared three different methods of analysing genotype data as a predictor of clinical response in a small clinical cohort of highly antiretroviral-experienced individuals failing therapy. No method performed well beyond 8 weeks into a new therapeutic regimen. A model based on the number of 'primary' mutations was statistically significant, but a multiple regression model, which identified specific mutations explained threefold more variation in response. Optimal prediction in this dataset was given by a model obtained from a classification tree analysis, in which genotype at amino acid sites 135 and 202 were combined with amino acid site 184, which explained over 50% of the deviance in the data and had a classification success of 86%.