Altered Levels of Decidual Immune Cell Subsets in Fetal Growth Restriction, Stillbirth, and Placental Pathology.

Immune cells are critically involved in placental development and functioning, and inadequate regulation of the maternal immune system is associated with placental pathology and pregnancy complications. This study aimed to explore numbers of decidual immune cells in pregnancies complicated with fetal growth restriction (FGR) and stillbirth (SB), and in placentas with histopathological lesions: maternal vascular malperfusion (MVM), fetal vascular malperfusion (FVM), delayed villous maturation (DVM), chorioamnionitis (CA), and villitis of unknown etiology (VUE). Placental tissue from FGR (n = 250), SB (n = 64), and healthy pregnancies (n = 42) was included. Histopathological lesions were classified according to criteria developed by the Amsterdam Placental Workshop Group. Tissue slides were stained for CD68 (macrophages), CD206 (M2-like macrophages), CD3 (T cells), FOXP3 [regulatory T (Treg) cells], and CD56 [natural killer (NK) cells]. Cell numbers were analyzed in the decidua basalis using computerized morphometry. The Mann-Whitney U-test and Kruskal Wallis test with the Dunn's as post-hoc test were used for statistical analysis. Numbers of CD68+ macrophages were higher in FGR compared to healthy pregnancies (p < 0.001), accompanied by lower CD206+/CD68+ ratios (p < 0.01). In addition, in FGR higher numbers of FOXP3+ Treg cells were seen (p < 0.01) with elevated FOXP3+/CD3+ ratios (p < 0.01). Similarly, in SB elevated FOXP3+ Treg cells were found (p < 0.05) with a higher FOXP3+/CD3+ ratio (p < 0.01). Furthermore, a trend toward higher numbers of CD68+ macrophages was found (p < 0.1) in SB. Numbers of CD3+ and FOXP3+ cells were higher in placentas with VUE compared to placentas without lesions (p < 0.01 and p < 0.001), accompanied by higher FOXP3+/CD3+ ratios (p < 0.01). Elevated numbers of macrophages with a lower M2/total macrophage ratio in FGR suggest a role for a macrophage surplus in its pathogenesis and could specifically indicate involvement of inflammatory macrophages. Higher numbers of FOXP3+ Treg cells with higher Treg/total T cell ratios in VUE may be associated with impaired maternal-fetal tolerance and a compensatory response of Treg cells. The abundant presence of placental lesions in the FGR and SB cohorts might explain the increase of Treg/total T cell ratios in these groups. More functionality studies of the observed altered immune cell subsets are needed.

Infant's Age Influences the Accuracy of Rectal Suction Biopsies for Diagnosing of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease (HD) is a rare birth defect of the distal colon. Analysis of rectal suction biopsy (RSB) is considered to be the most reliable method for its diagnosis in infants. However, the diagnostic accuracy of RSB analysis could be affected by the patient's age, possibly because of rapid development of the enteric nervous system in the first weeks after birth. Because there is a trend toward testing for HD at early ages, we aimed to determine whether the diagnostic accuracy of RSB analysis is associated with the patient's age. METHODS: We performed a retrospective analysis of all patients from whom 1 or more RSBs were analyzed from 1975 through 2011 (529 RSBs from 441 patients). Outcomes of RSB analyses were categorized as positive, inconclusive, or negative for HD. Primary diagnoses, based only on RSB, were compared with final diagnoses made after at least 1 year of clinical follow-up. Age at time of RSB analysis was corrected for the gestational age. By using these criteria, we determined the diagnostic accuracy of RSB analysis for different age groups. RESULTS: RSB analysis identified HD in patients with sensitivity values of 46% (patients -45 to 7 days old), 47% (8-22 days old), and 62% (23-39 days old) (corrected for gestational age). The average sensitivity with which RSB analysis identified HD in patients older than 39 days was 88%. RSB identified HD in patients younger than 39 days old with significantly lower sensitivity than in older patients (50% vs 88%, P < .001). The specificity with which RSB identified infants without HD was not affected by age (average 95%). Of all RSBs, 11% were inconclusive for the diagnosis of HD. CONCLUSIONS: RSB analysis identifies HD in patients younger than 39 days old with only 50% sensitivity. Moreover, RSBs obtained from younger patients often lead to inconclusive outcomes and require additional biopsies. We propose that for infants suspected of HD at these ages, a noninvasive technique, such as anorectal manometry, should be used for a primary diagnosis. RSB should thereafter be used to confirm the diagnosis when the infant is older than 39 days.

Cost-effectiveness of a screening strategy for Q fever among pregnant women in risk areas: a clustered randomized controlled trial.

BACKGROUND: In The Netherlands the largest human Q fever outbreak ever reported in the literature is currently ongoing with more than 2300 notified cases in 2009. Pregnant women are particularly at risk as Q fever during pregnancy may cause maternal and obstetric complications. Since the majority of infected pregnant women are asymptomatic, a screening strategy might be of great value to reduce Q fever related complications. We designed a trial to assess the (cost-)effectiveness of a screening program for Q fever in pregnant women living in risks areas in The Netherlands. METHODS/DESIGN: We will conduct a clustered randomized controlled trial in which primary care midwife centres in Q fever risk areas are randomized to recruit pregnant women for either the control group or the intervention group. In both groups a blood sample is taken around 20 weeks postmenstrual age. In the intervention group, this sample is immediately analyzed by indirect immunofluorescence assay for detection of IgG and IgM antibodies using a sensitive cut-off level of 1:32. In case of an active Q fever infection, antibiotic treatment is recommended and serological follow up is performed. In the control group, serum is frozen for analysis after delivery. The primary endpoint is a maternal (chronic Q fever or reactivation) or obstetric complication (low birth weight, preterm delivery or fetal death) in Q fever positive women. Secondary aims pertain to the course of infection in pregnant women, diagnostic accuracy of laboratory tests used for screening, histo-pathological abnormalities of the placenta of Q fever positive women, side effects of therapy, and costs. The analysis will be according to the intention-to-screen principle, and cost-effectiveness analysis will be performed by comparing the direct and indirect costs between the intervention and control group. DISCUSSION: With this study we aim to provide insight into the balance of risks of undetected and detected Q fever during pregnancy. TRIAL REGISTRATION: ClinicalTrials.gov, protocol record NL30340.042.09.

Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/EGP-2) in developing and adult kidneys.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed by most epithelia and is involved in processes fundamental for morphogenesis, including cell-cell adhesion, proliferation, differentiation, and migration. Previously, a role for EpCAM in pancreatic morphogenesis was confirmed in vitro. Furthermore, changes in the EpCAM expression pattern were found in developing lung and thymus and in the regenerating liver. Therefore, EpCAM was proposed to be a morphoregulatory molecule. METHODS: Using immunohistochemistry, the expression pattern of human and murine homologues of EpCAM was characterized in adult and embryonic kidneys from humans and human-EpCAM (hEpCAM)-transgenic mice. RESULTS: EpCAM expression was found in the ureteric bud throughout nephrogenesis. EpCAM was not expressed in the metanephric mesenchyme. In comma- and S-shaped bodies, both metanephric mesenchyme derived structures, EpCAM expression appeared by E13.5. In adult kidneys, most epithelia expressed varying levels of EpCAM, as confirmed by double staining for human EpCAM and segment-specific nephron markers. Podocytes were EpCAM negative. At the cellular level, the EpCAM expression shifted from apical in embryonic to basolateral in adult kidneys. CONCLUSIONS: The spatiotemporal expression pattern of EpCAM changes during nephrogenesis. In the adult kidney, the expression varies markedly along the nephron. These data provide a basis for further studies on EpCAM in developing and adult kidneys.

Plasma hemopexin activity in pregnancy and preeclampsia.

OBJECTIVE: Plasma hemopexin activity, associated with increased vascular permeability, was evaluated in healthy pregnant and non-pregnant women and in pre-eclamptic women. METHODS: Hemopexin activity and the hemopexin inhibitor, extracellular ATP, were assayed in plasma from pregnant (n = 10), preeclamptic (n = 9), and non-pregnant women (n = 10) using standard methods. Abdominal fascia tissue fragments from preeclamptic and pregnant women were immunohistochemically stained for vascular ecto-apyrase or ecto-5'nucleotidase. RESULTS: The data show significantly enhanced Hx activity exclusively in plasma from pregnant women and significantly enhanced plasma ATP in pre-eclamptic women compared with the other groups. Dephosphorylation of preeclamptic plasma resulted in reactivation of Hx activity. Fascia tissue-samples from preeclamptic women showed reduced ecto-apyrase activity and enhanced ecto-5'nucleotidase activity compared to pregnant women. CONCLUSION: Enhanced hemopexin activity may be associated with normal pregnancy, but not with preeclampsia. Decreased hemopexin in pre-eclamptic patients may be due to enhanced plasma ATP, which is possibly promoted by diminished activity of vascular ecto-apyrase.

Absence of in vivo generalized pro-inflammatory endothelial activation in severe, early-onset preeclampsia.

OBJECTIVE: At present it is unclear whether endothelial activation is systematically present in preeclampsia or restricted to specialized vascular beds. Therefore, this study aimed to investigate the presence of generalized proinflammatory endothelial activation in severe, early-onset preeclampsia in vivo. METHODS: During caesarean section, biopsies were obtained from abdominal subcutaneous fat, abdominal fascia, and myometrium from 11 severe, early-onset preeclamptic and 19 healthy pregnant women. Prior to caesarean, section plasma levels of von Willebrand Factor (vWF), sVCAM-1, and C-reactive protein (CRP) were measured by ELISA. Consecutive cryostat sections were stained immunohistochemically for CD31, E-selectin, VCAM-1, and ICAM-1. For subcutaneous fat tissue, endothelial gene expression levels of E-selectin, VCAM-1, ICAM-1, endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS) were quantified by real-time RT-PCR, using normalization to the endothelium-specific housekeeping genes CD31 and VE-cadherin. RESULTS: Plasma levels of vWF, sVCAM-1, and CRP were elevated in the preeclampsia group compared to the control group, indicating enhanced endothelial activation and inflammatory response in the severely diseased preeclamptic women. By immunohistochemical analysis, no E-selectin and VCAM-1 expression could be detected in, and no differences in endothelial ICAM-1 staining could be observed between the preeclampsia and the control group for all tissues studied. Endothelial gene expression levels of E-selectin, VCAM-1, ICAM-1, ET-1, and eNOS were comparable between the preeclampsia and control group. CONCLUSION: Protein and gene expression analysis of E-selectin, VCAM-1, ICAM-1, ET-1, and eNOS, key mediators involved in pro-inflammatory endothelial activation, could not identify endothelial activation in severe, early-onset preeclampsia in the tissues studied. However, elevated plasma levels of markers of endothelial activation and inflammation were observed. These results may suggest that in severe, early-onset preeclampsia pro-inflammatory endothelial cell activation is not a generalized phenomenon, but is likely restricted to (possibly organ-specific) specialized vascular beds.

Loss of desmoplakin tail causes lethal acantholytic epidermolysis bullosa.

The cytoplasmic plaque protein desmoplakin (DP), which is located in desmosomes, plays a major role in epithelial and muscle cell adhesion by linking the transmembrane cadherins to the cytoplasmic intermediate filament network. Mutations of DP may cause striate palmoplantar keratoderma, arrhythmogenic right ventricular dysplasia, skin fragility/woolly hair syndrome, Naxos-like disease, and Carvajal syndrome. DP must be indispensable, because DP-/- mice are early abortive. Here, we report a patient with severe fragility of skin and mucous membranes caused by genetic truncation of the DP tail. The new phenotype is lethal in the neonatal period because of immense transcutaneous fluid loss. The phenotype also comprised universal alopecia, neonatal teeth, and nail loss. Histology showed suprabasal clefting and acantholysis throughout the spinous layer, mimicking pemphigus. Electron microscopy revealed disconnection of keratin intermediate filaments from desmosomes. Immunofluorescence staining of DP showed a distinct punctate intercellular pattern in the patient's skin. Protein analysis revealed expression of truncated DP polypeptides. Mutational analysis of the patient demonstrated compound heterozygosity for two DP mutations, 6079C-->T (R1934X) and 6370delTT, respectively. Aberrant mRNA transcripts that predict premature termination of translation with loss of the three intermediate filament-binding subdomains in the DP tail were detected by RT-PCR. The new dramatic phenotype, which we named "lethal acantholytic epidermolysis bullosa," underscores the paramount role of DP in epidermal integrity.

Infantile and adult testicular germ cell tumors. a different pathogenesis?

Most adult testicular germ cell tumors have a characteristic chromosomal abnormality that is an isochromosome 12p [i(12p)]. Furthermore, these tumors are characterized by a chromosome number in the triploid range and gains and losses of (parts of) specific chromosomes. Cytogenetic investigation of three cases of infantile testicular germ cell tumors, all diagnosed as yolk sac tumors, revealed highly abnormal karyotypes. We found one case to be diploid; the other two cases were in the hypertriploid/hypotetraploid range. Structural abnormalities of chromosomes 1, 3, and 6 were recurrent and no i(12p) was found. Our results, together with data from the literature, suggest that infantile and adult testicular germ cell tumors have a different origin and pathogenetic pathway. Aberrations of chromosomes 1, 3, and 6 may play an important role in the pathogenesis of infantile testicular yolk sac tumors.