A Lassa virus mRNA vaccine confers protection but does not require neutralizing antibody in a guinea pig model of infection.

Lassa virus is a member of the Arenaviridae family, which causes human infections ranging from asymptomatic to severe hemorrhagic disease with a high case fatality rate. We have designed and generated lipid nanoparticle encapsulated, modified mRNA vaccines that encode for the wild-type Lassa virus strain Josiah glycoprotein complex or the prefusion stabilized conformation of the Lassa virus glycoprotein complex. Hartley guinea pigs were vaccinated with two 10 µg doses, 28 days apart, of either construct. Vaccination induced strong binding antibody responses, specific to the prefusion conformation of glycoprotein complex, which were significantly higher in the prefusion stabilized glycoprotein complex construct group and displayed strong Fc-mediated effects. However, Lassa virus-neutralizing antibody activity was detected in some but not all animals. Following the challenge with a lethal dose of the Lassa virus, all vaccinated animals were protected from death and severe disease. Although the definitive mechanism of protection is still unknown, and assessment of the cell-mediated immune response was not investigated in this study, these data demonstrate the promise of mRNA as a vaccine platform against the Lassa virus and that protection against Lassa virus can be achieved in the absence of virus-neutralizing antibodies.

Fetomaternal Hemorrhage following Placement of an Intrauterine Pressure Catheter: Report of a New Association.

Fetomaternal hemorrhage (FMH) can be associated with significant perinatal mortality. Our review of the literature did not identify any cases of FMH following placement of an intrauterine pressure catheter (IUPC). In our case, an IUPC was inserted in a patient undergoing induction of labor at term. Fetal bradycardia ensued shortly after placement, warranting an emergent cesarean delivery. Severe neonatal anemia was identified, and evaluation of maternal blood was consistent with massive FMH. This is the first reported association between FMH and IUPC placement. If this relationship is validated in future reports, appropriate changes in clinical practice may be warranted.

Complete hydatidiform mole and live fetus in a singleton pregnancy with confined placental mosaicism and fetomaternal hemorrhage: a case report.

BACKGROUND: Coexistence of complete mole and a live fetus is uncommon (1:22,000-100,000), more so with euploidy. CASE: We present a case of a molar pregnancy with a euploid fetus who had close fetal evaluation for second trimester bleeding. The patient presented at 29 weeks' pregnancy with decreased fetal movements, a result of fetomaternal hemorrhage. She underwent cesarean section and delivered a live infant. By close follow-up and a multidisciplinary approach, the appropriate diagnosis and a favorable outcome were achieved. Both mother and the child at 5 years of age are doing well. CONCLUSION: Detailed anatomic and molecular studies demonstrated a complete mole resulting from confined placental mosaicism, with molar tissue showing a single paternal allele at 8/8 informative loci, all shared with the fetus, thus this coexistent molar pregnancy was not that of a separate conceptus.

Placental release or disposal? Experiences of perinatal pathologists.

Placentas have been often considered medical waste in hospitals. This view is particularly held by the patients themselves, who may not understand the importance of placental examination. Hospitals have been receiving requests for placental release to patients and need to be prepared to handle these requests. Therefore, a survey was conducted to explore the experiences and practices of perinatal pathologists with respect to placental release. Utilizing SurveyMonkey, we emailed a survey to 192 practicing perinatal pathologists in the United States and Canada. Questions were asked about policies in force at their particular institution, conditions of release, and the purpose of release, ie, what the disposition of the placenta was after release to the family. Thirty-six responses were received; 22 (61.1%) of respondents did allow release of placentas, and those who did not release usually reported that they had not received requests for release. In most cases, specific policies were in place, with multiple departments within the hospital having input on the creation of the policy. Parental signature was required in most cases. The most common reason for patient request was to bury the placenta, although some placental release was for consumption and/or encapsulation. Although there are no specific religious requirements for use or burial of the placenta after delivery, there are many cultural reasons for requests. Hospitals and specific providers need to be aware of this interest and have a specific policy in place so that they are prepared when a request is received.

Molecular inversion probe array for the genetic evaluation of stillbirth using formalin-fixed, paraffin-embedded tissue.

Array comparative hybridization has been used successfully to identify genomic alterations in stillbirth material; however, high DNA quantity and quality requirements may limit its utility in some fetal samples. Molecular inversion probe (MIP) array analysis of FFPE stillbirth autopsy samples circumvents the challenges associated with karyotype and short-term fetal cell culture, requires limited DNA input, and allows for retrospective evaluation of fetal loss. We performed MIP analysis on archival FFPE autopsy tissue to identify underlying genetic abnormalities not previously detected using traditional cytogenetic methods. Archival FFPE stillbirth cases (≥20 weeks gestation) were identified with the following characteristics: i) the phenotype suggested underlying genomic alterations; ii) the karyotype was either normal or not available and there were no other known genetic abnormalities; or iii) previous microarray testing was not performed. Genomic DNA (75 ng) was processed onto a 330,000-feature MIP array. Twenty-seven of 29 (93.1%) FFPE samples had passing MIP quality control scores. Abnormalities were seen in 3 of 27 (11%) archival samples (deletion of 17q12, trisomy 18, and a case of 4qter duplication and 13qter deletion arising from an unbalanced 4q;13q translocation), which, if identified at the time of autopsy, may have changed the course of medical management. This study highlights the benefits of using MIP array analysis for identification of genomic alterations in FFPE stillbirth autopsy tissue.

Diagnostic utility of microsatellite genotyping for molar pregnancy testing.

CONTEXT: Molecular genotyping by analysis of DNA microsatellites, also known as short tandem repeats (STRs), is an established method for diagnosing and classifying hydatidiform mole. Distinction of both complete hydatidiform mole and partial hydatidiform mole from nonmolar specimens is relevant for clinical management owing to differences in risk for persistent gestational trophoblastic disease. OBJECTIVE: To determine the technical performance of microsatellite genotyping by using a commercially available multiplex assay, and to describe the application of additional methods to confirm other genetic abnormalities detected by the genotyping assay. DESIGN: Microsatellite genotyping data on 102 cases referred for molar pregnancy testing are presented. A separate panel of mini STR markers, flow cytometry, fluorescence in situ hybridization, and p57 immunohistochemistry were used to characterize cases with other incidental genetic abnormalities. RESULTS: Forty-eight cases were classified as hydatidiform mole (31, complete hydatidiform mole; 17, partial hydatidiform mole). Genotyping also revealed 11 cases of suspected trisomy and 1 case of androgenetic/biparental mosaicism. Trisomy for selected chromosomes (13, 16, 18, and 21) was confirmed in all cases by using a panel of mini STR markers. CONCLUSIONS: This series illustrates the utility of microsatellite genotyping as a stand-alone method for accurate classification of hydatidiform mole. Other genetic abnormalities may be detected by genotyping; confirmation of the suspected abnormality requires additional testing.

Cervical ribs are more prevalent in stillborn fetuses than in live-born infants and are strongly associated with fetal aneuploidy.

This study was undertaken to determine the prevalence of cervical ribs in stillborn fetuses undergoing autopsy at our institution and to search for significant associations with cervical ribs. European studies have reported an increased prevalence of cervical ribs in patients with childhood cancer and in stillborn fetuses. We reviewed data from autopsies performed at Primary Children's Medical Center, Utah, between 2006 and 2009 on 225 stillborns (≥20 weeks) and 93 deceased live-born infants (<1 year). Digital fetal radiographs in anterior-posterior and lateral views had been taken of each subject. Chi-square analysis and general linear models were used for statistical analysis of the data. The overall prevalence of cervical ribs was higher in stillborns than in live-borns who died in the first year (43.1% vs 11.8%). Karyotypes were available for 93 (41.3%) of the stillborns. Of those, cervical ribs were present in 33 of 76 (43.4%) stillborns with normal karyotype and in 13 of 17 (76.4%) stillborns with aneuploidy. Females with unavailable karyotypes were more likely to have cervical ribs than those with normal karyotypes (P  =  0.0002). This greater likelihood was not observed in males. Among the stillborns with normal karyotypes, we found no statistically significant association with gender or gestational age at fetal death. There was also no statistically significant association between congenital anomalies and the presence of cervical ribs. Our findings support the hypothesis that cervical ribs are markers for disadvantageous developmental events occurring during blastogenesis and have been subject to strong negative selection during evolution.

XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors.

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered in human prostate cancers and is the first gammaretrovirus known to infect humans. While gammaretroviruses have well-characterized oncogenic effects in animals, they have not been shown to cause human cancers. We provide experimental evidence that XMRV is indeed a gammaretrovirus with protein composition and particle ultrastructure highly similar to Moloney murine leukemia virus (MoMLV), another gammaretrovirus. We analyzed 334 consecutive prostate resection specimens, using a quantitative PCR assay and immunohistochemistry (IHC) with an anti-XMRV specific antiserum. We found XMRV DNA in 6% and XMRV protein expression in 23% of prostate cancers. XMRV proteins were expressed primarily in malignant epithelial cells, suggesting that retroviral infection may be directly linked to tumorigenesis. XMRV infection was associated with prostate cancer, especially higher-grade cancers. We found XMRV infection to be independent of a common polymorphism in the RNASEL gene, unlike results previously reported. This finding increases the population at risk for XMRV infection from only those homozygous for the RNASEL variant to all individuals. Our observations provide evidence for an association of XMRV with malignant cells and with more aggressive tumors.

Adenocarcinoma of the pancreas undetected by multidetector CT, endoscopic ultrasound, or intraoperative ultrasound.

CONTEXT: Patients with known or suspected pancreatic adenocarcinoma are typically evaluated with noninvasive imaging studies and endoscopic ultrasound. Rarely, patients require intraoperative evaluation with intraoperative ultrasound to identify mass lesions. Some patients have pancreatic adenocarcinomas that cannot be detected using any of these methods. CASE REPORT: A-58-year old female presented with a distal common bile duct stricture seen on ERCP with negative brushings. Multiple endoscopic ultrasound and triple phase pancreatic protocol CT exams were negative for a mass lesion and revealed a normal pancreas. Intraoperative ultrasound of the pancreas was also felt to be normal. Intraoperative biopsy of the head of the pancreas revealed a small, moderately to poorly differentiated adenocarcinoma, not visible on any of her imaging studies. CONCLUSION: Some pancreatic adenocarcinomas may defy detection using modern imaging modalities. This case illustrates how extensive imaging failed to detect a malignancy prior to surgery. Patients with a high clinical suspicion for malignancy but no visualized mass should undergo operative evaluation with definitive tissue sampling.

Placental biomarkers of phthalate effects on mRNA transcription: application in epidemiologic research.

BACKGROUND: CYP19 and PPARgamma are two genes expressed in the placental trophoblast that are important to placental function and are disrupted by phthalate exposure in other cell types. Measurement of the mRNA of these two genes in human placental tissue by quantitative real-time polymerase chain reaction (qPCR) offers a source of potential biomarkers for use in epidemiologic research. We report on methodologic challenges to be considered in study design. METHODS: We anonymously collected 10 full-term placentas and, for each, sampled placental villi at 12 sites in the chorionic plate representing the inner (closer to the cord insertion site) and outer regions. Each sample was analyzed for the expression of two candidate genes, aromatase (CYP19) and peroxisome proliferator activated receptor protein gamma (PPARgamma) and three potential internal controls: cyclophilin (CYC), 18S rRNA (18S), and total RNA. Between and within placenta variability was estimated using variance component analysis. Associations of expression levels with sampling characteristics were estimated using mixed effects models. RESULTS: We identified large within-placenta variability in both transcripts (>90% of total variance) that was minimized to <20% of total variance by using 18S as an internal control and by modelling the means by inner and outer regions. 18S rRNA was the most appropriate internal control based on within and between placenta variability estimates and low correlations of 18S mRNA with target gene mRNA. Gene expression did not differ significantly by delivery method. We observed decreases in the expression of both transcripts over the 25 minute period after delivery (CYP19 p-value for trend = 0.009 and PPARgamma (p-value for trend = 0.002). Using histologic methods, we confirmed that our samples were comprised predominantly of villous tissue of the fetal placenta with minimal contamination of maternally derived cell types. CONCLUSION: qPCR-derived biomarkers of placental CYP19 and PPARgamma gene expression show high within-placental variability. Sampling scheme, selection of an appropriate internal control and the timing of sample collection relative to delivery can be optimized to minimize within-placenta and other sources of underlying, non-etiologic variability.

Soluble Ig-like transcript 3 inhibits tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients.

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

False-positive quadruple screen test for trisomy 18 in a patient with a fetus with Bloom's syndrome.

In the literature, conflicting reports on the significance of false-positive maternal serum multiple marker testing for trisomy 18 are encountered; however, the biology of this finding is discussed infrequently. We present such a case in association with Bloom's syndrome in the fetus. The fetus had intrauterine growth restriction, seen early in the second trimester, oligohydramnios, and was delivered at 34 weeks of gestation for impending fetal compromise. We propose that the adverse outcome of the pregnancy with false-positive serum analyte testing for trisomy 18 might result from a small-sized placenta and perhaps pathology at receptor level.

Antenatal sonographic prediction of twin chorionicity.

OBJECTIVE: We sought to determine the accuracy of antenatal diagnosis of twin chorionicity at a single tertiary care center and assess the consequences of incorrect diagnoses. STUDY DESIGN: Twins with chorionicity diagnosed by ultrasound < or = 24 weeks' gestation were retrospectively reviewed. Chorionicity was assigned by sonographic findings including placental location(s), the lambda and T-signs, and/or fetal gender(s). Postnatal diagnosis was determined by placental histopathologic examination. Medical records of antenatal-postnatal discordant chorionicities were reviewed for adverse sequelae. RESULTS: Chorionicity was correctly assigned antenatally in 392/410 (95.6%) twins. The sensitivity, specificity, and positive and negative predictive values of monochorionicity assessed < or = 14 weeks were 89.8%, 99.5%, 97.8%, and 97.5%. Corresponding statistical values for the second trimester were 88.0%, 94.7%, 88.0%, and 94.7%. Two cases of inaccurate antenatal diagnoses affected patient counseling or were associated with adverse clinical outcomes. CONCLUSION: Antenatal assessment of chorionicity is accurate; however, incorrect diagnoses do occur and can affect reliable patient counseling and management.

Ascending aortic aneurysm in a fetus due to a benign nodular myofibroblastic lesion.

A fetal echocardiogram at 20 weeks of gestation revealed a large ascending aortic aneurysm in the presence of a normal aortic root and normal intracardiac anatomy. No other abnormalities were noted in the fetus. Upon termination of pregnancy, histopathological examination revealed an isolated benign nodular myofibroblastic lesion of likely hamartomatous origin, a first description of such pathology contributing to the formation of an aneurysm in the ascending aorta.

Prenatal diagnosis of glycogen storage disease type IV.

BACKGROUND: Glycogen storage disease type IV (GSD-IV) is a rare autosomal recessive disorder due to mutations in the GBE1 gene causing deficiency of the glycogen branching enzyme (GBE). Prenatal diagnosis has occasionally been performed by the measurement of the GBE activity in cultured chorionic villi (CV) cells. METHODS: Two unrelated probands with severe hypotonia at birth and death during the neonatal period were diagnosed with GSD-IV on the basis of postmortem histological findings. DNA analysis revealed truncating GBE1 mutations in both families. RESULTS: Prenatal diagnosis was performed in subsequent pregnancies by determination of branching enzyme activity and DNA analysis of CV or cultured amniocytes. Detailed autopsies of the affected fetuses at 14 and 24 weeks of gestation demonstrated intracellular inclusions of abnormal glycogen characteristic of GSD-IV. CONCLUSION: Prenatal diagnosis of GSD-IV by DNA analysis is highly accurate in genetically confirmed cases.

Alpha-thalassemia major presenting in a term neonate without hydrops.

We describe the clinical and pathologic features of an unusual case of alpha-thalassemia major in a patient who survived to term and lived for 9 days. The neonate was nonhydropic and the clinical picture was dominated by severe hypoxia with pulmonary hypertension. The diagnosis was not suspected until postnatal examination of the blood smear, which prompted the performance of hemoglobin electrophoresis and subsequent molecular confirmation. This case illustrates that alpha-thalassemia major should be in the differential diagnosis of hypoxic neonates even in the absence of hydrops.

Immunohistochemistry for the imprinted gene product IPL/PHLDA2 for facilitating the differential diagnosis of complete hydatidiform mole.

OBJECTIVE: To determine if immunohistochemistry for PHLDA2 (also known as IPL and TSSC3), the product of a paternally imprinted, maternally expressed gene, can be used as a tool in the differential diagnosis of molar gestations. STUDY DESIGN: Twenty-five cases (15 complete moles, 5 partial moles and five hydropic abortions) were stained by immunohistochemistry for PHLDA2 and scored (without knowledge of the diagnosis) for positivity in the villous cytotrophoblast and then compared to adjacent sections stained by p57KIP2 immunohistochemistry. RESULTS: All partial moles and hydropic abortions were positive for PHLDA2 and p57KIP2. There was strong PHLDA2 staining of the cytoplasm in virtually all cells of the villous cytotrophoblast, while p57KIP2 was localized to the nucleus in a subset of those cells. All complete moles were negative for both markers in the villous cytotrophoblast. CONCLUSION: Immunohistochemistry for PHLDA2 serves as a practical and reliable diagnostic marker for the discrimination of complete mole from partial mole and hydropic abortion. Since the immunohistochemical diagnosis of complete mole is based on a negative result, absence of staining, the use of both markers (PHLDA2 and p57KIP2) together could increase the level of confidence when making this prognostically important distinction.