Identification of diabetic retinopathy genes through a genome-wide association study among Mexican-Americans from Starr County, Texas

To identify genetic susceptibility loci for severe diabetic retinopathy, 286 Mexican-Americans with type 2 diabetes from Starr County, Texas completed detailed physical and ophthalmologic examinations including fundus photography for diabetic retinopathy grading. 103 individuals with moderate-to-severe non-proliferative diabetic retinopathy or proliferative diabetic retinopathy were defined as cases for this study. DNA samples extracted from study subjects were genotyped using the Affymetrix GeneChip® Human Mapping 100K Set, which includes 116,204 single nucleotide polymorphisms (SNPs) across the whole genome. Single-marker allelic tests and 2- to 8-SNP sliding-window Haplotype Trend Regression implemented in HelixTreeTM were first performed with these direct genotypes to identify genes/regions contributing to the risk of severe diabetic retinopathy. An additional 1,885,781 HapMap Phase II SNPs were imputed from the direct genotypes to expand the genomic coverage for a more detailed exploration of genetic susceptibility to diabetic retinopathy. The average estimated allelic dosage and imputed genotypes with the highest posterior probabilities were subsequently analyzed for associations using logistic regression and Fisher's Exact allelic tests, respectively. To move beyond these SNP-based approaches, 104,572 directly genotyped and 333,375 well-imputed SNPs were used to construct genetic distance matrices based on 262 retinopathy candidate genes and their 112 related biological pathways. Multivariate distance matrix regression was then used to test hypotheses with genes and pathways as the units of inference in the context of susceptibility to diabetic retinopathy. This study provides a framework for genome-wide association analyses, and implicated several genes involved in the regulation of oxidative stress, inflammatory processes, histidine metabolism, and pancreatic cancer pathways associated with severe diabetic retinopathy. Many of these loci have not previously been implicated in either diabetic retinopathy or diabetes. In summary, CDC73, IL12RB2, and SULF1 had the best evidence as candidates to influence diabetic retinopathy, possibly through novel biological mechanisms related to VEGF-mediated signaling pathway or inflammatory processes. While this study uncovered some genes for diabetic retinopathy, a comprehensive picture of the genetic architecture of diabetic retinopathy has not yet been achieved. Once fully understood, the genetics and biology of diabetic retinopathy will contribute to better strategies for diagnosis, treatment and prevention of this disease.^

Characterization of Staphylococcus aureus isolates associated with pediatric carriage in Houston, Texas

Infections caused by Methicillin-resistant Staphylococcus aureus (MRSA) have been of great concern in hospitals due the difficulty in treating virulent, antibiotic resistant microorganisms in sensitive populations including children, the elderly, and immunocomprimised individuals. Since the late 1990's, MRSA infections have become a problem in the general community, and the strains of S. aureus that cause infections in the community are known to be genetically different than the hospital acquired strains. Community-acquired strains tend to be more virulent, affecting even relatively healthy individuals, and disease presentation tends to be more diverse than diseases observed in patients suffering from hospital-acquired strains. From the year 2000 to the present, there has been a significant increase in community-acquired infections in children, a population already particularly sensitive to S. aureus infection. Genotyping the strains of CA-MRSA circulating in the pediatric population is an important step in developing better antibiotic treatment strategies. Additionally, determining the carriage status of individuals in this population and comparing these data with strain genotypes will also be valuable in establishing prevention and control practices. ^

Association of pellicle growth morphological characteristics and clinical presentation of Mycobacterium tuberculosis isolates

Trehalose dimycolate (TDM) is a mycobacterial glycolipid that is released from the surface of virulent M. tuberculosis. We evaluated the rate of growth, colony characteristics and production of TDM by Mycobacterium tuberculosis strains isolated from different clinical sites. Since detergent removes TDM from organisms, we analyzed growth rate and colony morphology of 79 primary clinical isolates grown as pellicles on the surface of detergent free Middlebrook 7H9 media. The genotype of each had been previously characterized. TDM production was measured by thin layer chromatography on 32 of these isolates. We found that strains isolated from pulmonary sites produced large amounts of TDM, grew rapidly as thin spreading pellicles, showed early cording (<1 week) and climbed the sides of the dish. In contrast, the extrapulmonary isolates (lymph node and bone marrow) produced less TDM (p<0.01), grew as discrete patches with little tendency to spread or climb the walls (p<0.02). The Beijing pulmonary (BP) isolates produced more TDM than non Beijing pulmonary isolates. The largest differences were observed in Beijing strains. The Beijing pulmonary isolates produced more TDM and grew faster than the Beijing extrapulmonary isolates (p<0.01). This was true even when the pulmonary and extrapulmonary isolates were derived from the same clade. These growth characteristics were consistently observed only on the first passage after primary isolation. This suggests that the differences in growth rate and TDM production observed reflect differences in gene expression patterns of pulmonary and extrapulmonary infections, that Mycobacterium tuberculosis in the lung grows more rapidly and produces more TDM than it does in extrapulmonary sites. This provides new opportunities to investigate gene expression of Mycobacterium tuberculosis in human.^

Modulation of radiation-induced genetic damage by HCMV: A glioma case-control study

Human cytomegalovirus (HCMV) infection occurs early in life and leads to life-long viral persistence. An association between HCMV infection and malignant gliomas has been reported suggesting that HCMV may play a role in glioma pathogenesis. The reported effects of HCMV on cells suggest that it could facilitate accrual of genotoxic damage. We therefore tested the hypothesis that HCMV infection modifies the sensitivity of cells to genetic damage from environmental insults such as γ-irradiation. Peripheral blood lymphocytes from 110 glioma patients and 100 controls were used to measure the level of both chromosome damage and cell death as endpoints for genetic instability. For each study participant, the extent of baseline, HCMV-, γ-radiation- and both – induced genetic instability was evaluated. Radiation induced a significant increase in aberration frequency over baseline in both cases and controls. Similarly, HCMV induced a significant increase in aberration frequency regardless of the disease status. Interestingly, HCMV induced damage was either equal or higher than that induced by radiation. Infected with HCMV prior to challenge with γ-radiation demonstrated a significant increase in the aberration frequency as compared to baseline, radiation- or HCMV-treated cells. With regards to apoptosis, cases showed a lower percentage of induction following in vitro exposure to γ-radiation and/or HCMV infection. The level of apoptosis was inversely related to the amount of chromosome damage in the cases, but not in the controls. These data indicate that, HCMV infection enhances the sensitivity of PBLs to γ-radiation-induced genetic damage.^

Assessing pediatric nasal carriage of S. aureus & MRSA

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) has become an increasing problem in the community. Nasal carriage of these bacteria has been shown to be a predisposing factor for infection and environmental contamination. This serious public health concern prompted an investigation to assess the pediatric nasal carriage of these bacteria in an effort to better understand the populations at risk and prevention of infection.^ This prospective study surveys 30 children from the Northwest Assistance Ministries (NAM) pediatric clinic from October 2008 to the present. Two nasal swabs were taken in 2-4 week intervals to determine S. aureus carrier status. Microbiologic tests were conducted to isolate and identify S. aureus from nasal cultures. Children with 2 cultures positive for S. aureus were classified as persistent carriers, those with 1 positive and 1 negative culture were classified as intermittent carriers, and those with 2 negative cultures were classified as noncarriers. This information was related to patient records and statistical analyses (X 2 and t-tests) were performed.^ Distribution of S. aureus carriage related to patient demographics (age, sex, & race) was showed no significant differences between S. aureus positive and S. aureus negative patient populations (p = 0.8). Additionally, the distribution of carrier status related to demographics also showed no significant difference (p = 0.8). Finally, the distribution of carrier status related to relevant medical history (immunizations current, past infection, & antibiotic use at time of swabbing) showed no significant difference (p = 0.4).^ This study is a snapshot of an ongoing study to assess the pediatric nasal carriage of S. aureus and MRSA. The inability to draw any reliable conclusion from the distribution of data is likely a result of an inadequate samples size. This is one of few studies assessing pediatric nasal carriage of S. aureus and targeting an underrepresented, Hispanic population is especially unique. Continuing this study allows for a better understanding of the epidemiology of this bacterium which will hopefully lead to appropriate interventions thus preventing future S. aureus infections.^

Matrix metalloproteinase-1 as a prognosticator in malignant glioma

The overall purpose of this study was to assess the relationship between the promoter region polymorphism (-2607 1G/2G) of matrix metalloproteinase-1 (MMP-1) polymorphism and outcome in brain tumor patients diagnosed with a primary brain tumor between 1994 and 2000 at The University of Texas M. D. Anderson Cancer Center. The MMP-1 polymorphism was genotyped for all brain tumor patients who participated in the Family Brain Tumor Study and for whom blood samples were available. Relevant covariates were abstracted from medical records for all cases from the original protocol, including information on demographics, tumor histology, therapy and outcome was obtained. The hypothesis was that brain tumor patients with the 2G allele have a poorer prognosis and shorter survival than brain tumor patients with the 1G allele. ^ Experimental Design: Genetic variants for the MMP-1 enzyme were determined by a polymerase chain reaction-restriction fragment length polymorphism assay. Comparison was made between the overall survival for cases with the 2G polymorphism and overall survival for cases with the 1G polymorphism using multivariable Cox Proportional-Hazard analysis, controlling for age, sex, Karnofsky Performance Scale (KPS), extent of surgery, tumor histology and treatment received. Kaplan-Meier and Cox Proportional-Hazard analyses were utilized to assess if the MMP-1 polymorphisms were related to overall survival. Results: Overall survival was not statistically significantly different between the 2G allele brain tumor patients and the 1G allele patients and there was no statistically significant difference between tumor types. ^ Conclusions: No association was found between MMP-1 polymorphisms and survival in patients with malignant gliomas. ^

Ethnic differences in tumor characteristics, treatment and survival in glioma patients

Purpose. A descriptive analysis of glioma patients by race was carried out in order to better elucidate potential differences between races in demographics, treatment, characteristics, prognosis and survival. ^ Patients and Methods. Among 1,967 patients ≥ 18 years diagnosed with glioma seen between July 2000 and September 2006 at The University of Texas M.D. Anderson Cancer Center (UTMDACC). Data were collated from the UTMDACC Patient History Database (PHDB) and the UTMDACC Tumor Registry Database (TRDB). Chi-square analysis, uni- /multivariate Cox proportional hazards modeling and survival analysis were used to analyze differences by race. ^ Results. Demographic, treatment and histologic differences exist between races. Though risk differences were seen between races, race was not found to be a significant predictor in multivariate regression analysis after accounting for age, surgery, chemotherapy, radiation, tumor type as stratified by WHO tumor grade. Age was the most consistent predictor in risk for death. Overall survival by race was significantly different (p=0.0049) only in low-grade gliomas after adjustment for age although survival differences were very slight. ^ Conclusion. Among this cohort of glioma patients, age was the strongest predictor for survival. It is likely that survival is more influenced by age, time to treatment, tumor grade and surgical expertise rather than racial differences. However, age at diagnosis, gender ratios, histology and history of cancer differed significantly between race and genetic differences to this effect cannot be excluded. ^

Aedes aegypti, Aedes albopictus, and Dengue virus in Harris county: An estimate of risk

Recent outbreaks of dengue fever (DF) along the United States/Mexico border, coupled with the high number of reported cases in Mexico suggest that there is the possibility for DF emergence in Houston, Texas1,2. To determine the presence of DF, populations of Aedes aegypti and Aedes albopictus were identified and tested for dengue virus. Maps were created to identify "hot spots" (Figure 1) based on historical data on Ae. aegypti and Ae. albopictus, demographic information, and locations of human cases of dengue fever. BG Sentinel Traps®, in conjunction with BG Lure® attractant, octanol and dry ice, were used to collect mosquitoes, which were then tested for presence of dengue virus using ELISA techniques. All samples tested were negative for dengue virus (DV). Survival of DV ultimately comes down to whether or not it will be vectored by a mosquito to a susceptible human host. The presence of infected humans and contact with the mosquito vectors are two critical factors necessary in the establishment of DF. Historical records indicate the presence of Ae. aegypti and Ae. albopictus in Harris County, which would support localized dengue transmission if infected individuals are present.^ (1) Brunkard JM, Robles-Lopez JL, Ramirez J, Cifuentes E, Rothenberg SJ, Hunsperger EA, Moore CG, Brussolo RM, Villarreal NA, Haddad BM, 2007. Dengue fever seroprevalence and risk factors, Texas-Mexico border, 2004. Emerg Infect Dis 13: 1477-1483. (2) Ramos MM, Mohammed H, Zielinski-Gutierrez E, Hayden MH, Lopez JL, Fournier M, Trujillo AR, Burton R, Brunkard JM, Anaya-Lopez L, Banicki AA, Morales PK, Smith B, Munoz JL, Waterman SH, 2008. Epidemic dengue and dengue hemorrhagic fever at the Texas-Mexico Border: results of a household-based seroepidemiologic survey, December 2005. Am J Trop Med Hyg 78: 364-369.^

Systematic review of genetic risk score in coronary heart disease and other diseases

In order to better take advantage of the abundant results from large-scale genomic association studies, investigators are turning to a genetic risk score (GRS) method in order to combine the information from common modest-effect risk alleles into an efficient risk assessment statistic. The statistical properties of these GRSs are poorly understood. As a first step toward a better understanding of GRSs, a systematic analysis of recent investigations using a GRS was undertaken. GRS studies were searched in the areas of coronary heart disease (CHD), cancer, and other common diseases using bibliographic databases and by hand-searching reference lists and journals. Twenty-one independent case-control studies, cohort studies, and simulation studies (12 in CHD, 9 in other diseases) were identified. The underlying statistical assumptions of the GRS using the experience of the Framingham risk score were investigated. Improvements in the construction of a GRS guided by the concept of composite indicators are discussed. The GRS will be a promising risk assessment tool to improve prediction and diagnosis of common diseases.^

Regulation of two P450 isoforms: CYP2D6 and CYP4F11

Cytochromes P450 catalyze a monooxygenase reaction in which molecular oxygen is split and one oxygen atom is incorporated into the substrate. As a whole, P450 researchers have focused most of their attention on substrate metabolism and relatively little on how these enzymes are regulated. This study will focus on the regulation of two P450 isoforms known as, CYP2D6 and CYP4F11. ^ The human CYP2D gene locus contains two pseudogenes and one functional gene known as CYP2D6. This locus is highly polymorphic and produces several alternatively spliced transcripts from the pseudogene CYP2D7. My objective was to understand the role of SV5-in (splice variant 5), one of several alternative splice variants transcribed from the CYP2D7 pseudogene. My results indicate that SV5-in mRNA causes an increase in CYP2D6 protein levels and suggest that there is a role for SV5-in in regulation of CYP2D6 expression. ^ Second, CYP4F11 is a recently discovered and uncharacterized isoform, derived from the CYP4F subfamily. It metabolizes several clinically relevant drugs (i.e.—erythromycin and benzphetamine) and some endogenous inflammatory mediators (i.e.—LTB4). After evaluation of microarray data, I observed an increase in CYP4F11 mRNA levels from wild-type HCT116 cells compared to p53-null cells. Our objectives were to explore and understand this connection between p53 and CYP4F11. Microarray data were confirmed by Q-PCR, after which this effect was again observed at the protein level via Western blot and again at the promoter level via luciferase assay and chromatin immunoprecipitation. Our results indicate that p53 protein regulates expression of CYP4F11 mRNA and protein through CYP4F11 promoter binding (note that p53 binding to CYP4F11 DNA was not shown to be direct). These results signify a whole new level of regulation of drug metabolizing enzymes by p53. ^ An understanding of CYP4F11 regulation by p53 could help us understand another pathway leading to apoptosis or cell growth arrest. This can aid future drug studies and discover new drug metabolism pathways under the control of a tumor suppressor protein. An understanding of the CYP2D6 regulation pathway could illuminate the role of non-coding RNAs in the P450 field and potentially explain several inter-individual drug response variations observed in clinical medicine that are not yet completely explained by genotyping analysis. ^

Organization and function of platelet glycoprotein Ib-IX complex

Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits—GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function. ^

NHERF1 recruits the tumor suppressors PTEN and PHLPP to synergistically inhibit the PI-3K pathway

Glioblastoma multiforme is the most common form of brain cancer that presents patients with a poor prognosis that has remained unchanged over the past few decades. The tumor suppressor phosphatase PTEN antagonizes one of the major oncogenic pathways involved in the progression of glioblastoma, and is frequently deleted in this cancer type. Contrary to our expectations, we found that most glioblastoma cells expressing endogenous PTEN also harbor basal PI-3K/AKT activation mainly attributable to impaired PTEN membrane localization. This alteration correlated with a shift of the adaptor protein NHERF1, which contributes to PTEN membrane recruitment in normal cells, from the membrane to the cytoplasm. In cells expressing membrane-localized NHERF1, only simultaneous PTEN and NHERF1 depletion achieved AKT activation, suggesting the involvement of additional PI-3K/AKT suppressor regulated by NHERF1. We identified these novel interactors of NHERF1 as the PHLPP1 and PHLPP2 phosphatases. ^ NHERF1 directly interacted and recruited both PHLPP proteins to the membrane and, through both NHERF1 PDZ domains, assembled ternary complexes consisting of PTEN-NHERF1-PHLPP. Only simultaneous depletion of PTEN and PHLPP1 significantly activated AKT and increased proliferation in cells with membrane-localized NHERF1. Analysis of glioblastoma human tumors revealed frequent loss of membrane-localized NHERF1. On the other hand, targeting of NHERF1 to the membrane achieved suppression of AKT and cell proliferation. Our findings reveal a novel mechanism for PI-3K/AKT regulation by the synergistic cooperation between two important tumor suppressors, PTEN and PHLPP, via the scaffold protein NHERF1. ^

A systematic analysis of INO80 in DNA replication

ATP-dependent chromatin remodeling has been shown to be critical for transcription and DNA repair. However, the involvement of ATP-dependent chromatin remodeling in DNA replication remains poorly defined. Interestingly, we found that the INO80 chromatin-remodeling complex is directly involved in the DNA damage tolerance pathways activated during DNA replication. DNA damage tolerance is important for genomic stability and is controlled by formation of either mono-ubiquitinated or multi-ubiquitinated PCNA, which respectively induce error prone or error-free replication bypass of the lesions. In addition, homologous recombination (HR) mediated by the Rad51 pathway is also involved in the DNA damage tolerance pathways. ^ We found that INO80 is specifically recruited to replication origins during S phase in a genome-wide fashion. In addition, DNA combing analysis shows INO80 is required for the resumption of replication at stalled forks induced by methyl methane-sulfonate (MMS). Mechanistically, we find that INO80 is required for PCNA ubiquitination as well as for Rad51 mediated processing of replication forks after MMS treatment. Furthermore, chromatin immunoprecipitation at specific ARSs indicates INO80 is necessary for Rad18 and Rad51 recruitment to replication forks after MMS treatment. Moreover, 2D gel analysis shows INO80 is necessary to process Rad51 mediated intermediates at impeded replication forks. ^ In conclusion, our findings establish a novel role of a chromatin-remodeling complex in DNA damage tolerance pathways and suggest that chromatin remodeling is fundamentally important to ensure faithful replication of DNA and genome stability in eukaryotes. ^

Establishment of a complex inflammatory and protumorigenic microenvironment in mouse pancreas through cyclooxygenase-2 overexpression

Chronic inflammation is an established risk factor in the pathogenesis of many cancers. Pancreatic ductal adenocarcinoma, a malignancy with a particularly dismal prognosis, is no exception. Cyclooxygenase-2, a key enzyme induced by tissue injury, has a critical role in the generation of bioactive lipids known as prostaglandins. COX-2 overexpression is a frequent finding in pancreatic cancer, chronic pancreatitis and pancreatic intraepithelial neoplasias. To explore mechanisms through which chronic inflammation establishes and maintains a protumorigenic environment, we designed a mouse model overexpressing COX-2 in pancreatic parenchyma (BK5.COX-2 mice). We discovered that constitutive expression of COX-2 has a number of important sequelae, including upregulation of additional eicosanoid-generating enzymes and proinflammatory cytokines. Many of these molecular alterations precede the onset of significant histopathological changes. Increased levels of prostaglandins E2, D2, and F2α, 5-, 12-, and 15-hydroxyeiosatetraenoic acid (HETEs) were documented in tumors and pancreata of younger transgenic mice. Using a TaqMan™ Mouse Immune Panel, we detected elevated mRNAs for a number of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6). ^ Histological examination revealed early changes in the pancreas with similarities to human chronic pancreatitis, including loss of acinar cells, appearance of metaplastic ducts, and increased deposition of stroma. As the lesions progress, features typical of dysplastic and neoplastic cells emerged within the metaplastic ductal complexes, including cellular and nuclear atypia, crowding of cells, and loss of normal tissue architecture. The amount of fibroinflammatory stroma increased considerably; numerous small vessels were evident. A number of immunocytes from both the myeloid and lymphoid lineages were identified in transgenic pancreata. Neutrophils were the earliest to infiltrate, followed shortly by macrophages and mast cells. B and T cells generally began to appear by 8–12 weeks, and organized aggregates of lymphoid cells were often found in advanced lesions. ^ We tested the efficacy of several chemopreventive agents in this model, including celecoxib, a COX-2 selective inhibitor, pentoxifylline, a cytokine inhibitor, curcumin, a polyphenol with antioxidant and anti-inflammatory properties, and GW2974, a dual EGFR/ErbB2 inhibitor. Effects on lesion development were modest in the GW2974 and pentoxifylline treated groups, but significant prevention effects were observed with curcumin and celecoxib. ^

Seroprevalence of toxoplasmosis and current thinking regarding screening in pregnancy: A review of the literature and case study discussion

The approach to the diagnosis and treatment of congenital toxoplasmosis has been one of flux and debate, fueled by lack of knowledge, lack of consensus, different methods of screening and different national policies for screening in different parts of the world. Countries with higher prevalence of disease such as in Europe and South America have a heightened awareness of the need to screen and treat for this parasitic infection during pregnancy. In contrast, in the United States, it is a condition scarcely discussed and has been largely ignored except in some large centers and by a few researchers. Policies and research strategies for any condition should start with obtaining good data. The aims of this thesis included a review of prevalence studies conducted in the United States, focused on the past 20 years, combined with a description of original research conducted by the author several years ago. The latter was a cross-sectional study performed in Houston, one of the largest American cities with a great ethnic mix. The study analyzed prevalence rates of Toxoplasma gondii IgG antibody in sera of women of reproductive age. Overall seroprevalence was 12.3%. In keeping with other studies, higher prevalence correlated with lower socioeconomic status, Black and Hispanic and Asian ethnicities, and increasing age. A literature search revealed only three prevalence studies performed in the United States over the past 20 years, with another four studies only referred to as personal communications or within a textbook, without further study detail available. The literature review also revealed a lack of consensus on whether or not to screen for toxoplasmosis in pregnancy, and even whether or not treatment in utero is worthwhile.^ Proponents of screening and treatment in pregnancy site studies both in the United States and France, emphasize that treatment reduces disease manifestations in infants. Opponents cite other studies that show only marginal benefits, together with potential side effects of medication regimens and generation of anxiety in parents. What is agreed on so far is the value of educating pregnant women on how to avoid contracting toxoplasmosis, and educating physicians on making the best use of reference laboratories before major treatment decisions are made. Further research to reevaluate the literature critically, review new treatment regimens and examine costs and benefits of screening and treatment of toxoplasmosis in pregnancy, bringing together European and American researchers, is needed.^