William K. Rashbaum

Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block

An important advance in the description and understanding of congenital heart block (CHB) came in the 1970s with the observation that mothers of affected infants frequently had autoimmune diseases and, in particular, that many maternal sera contained antibodies to SSA/Ro and SSB/La ribonucleoproteins. Although the molecular biology of the candidate antigens has been extensively defined, the arrhythmogenic and electrophysiological effects of their cognate antibodies on the human fetal heart are unknown. In the present study, we provide evidence that IgG-enriched fractions and anti-52-kD SSA/Ro antibodies affinity-purified from sera of mothers whose children have CHB induce complete atrioventricular (AV) block in the human fetal heart perfused by the Langendorff technique and inhibit L-type Ca2+ currents at the whole-cell and single-channel level. Immunization of female BALB/c mice with recombinant 52-kD SSA/Ro protein generated high-titer antibodies that crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities, including complete AV block, in the pups. These findings strongly suggest that anti-52-kD SSA/Ro antibodies are causally related to the development of CHB.

Human fetal astrocytes induce the expression of blood-brain barrier specific proteins by autologous endothelial cells

The blood-brain barrier (BBB) is involved in many normal regulatory mechanisms as well as in pathologic conditions of the central nervous system. Previous studies examining the development and function of the BBB in vitro have primarily utilized cell lines or cultured tissues from non-human sources. In contrast, this study used a coculture system of human fetal astrocytes and autologous endothelial cells. Astrocytes and endothelial cells (EC) were isolated and cultured on the opposite sides of a synthetic permeable membrane. The cocultures were characterized by electron and light microscopy for morphology and by immunocytochemistry for cell-type specific markers. Using these coculture conditions, astrocytes displayed characteristic morphology and expressed glial fibrillary acidic protein. When cocultured with astrocytes, endothelial cells retained factor VIII expression and expressed the BBB-specific proteins, brain-type glucose transporter (GLUT-1) and gamma-glutamyl transpeptidase. This expression was dependent on EC being in close apposition to or in direct contact with astrocytes. The model presented in this study may permit further examination of the role of the BBB in both normal human neurodevelopment and neuropathologic conditions.

Detection of HIV in fetal central nervous system tissue

Neurological disease is a common finding in children with AIDS and in others without signs of disease but with evidence of congenital HIV-1 infection. To investigate the possibility that HIV-1 can infect fetal central nervous system (CNS) tissue and therefore possibly serve as the substrate for the abnormal neurodevelopment characteristic of pediatric AIDS, eight abortus CNS samples (one set of twins) from seven HIV-1-seropositive intravenous drug users (IVDUs) and eight control abortus CNS samples from eight HIV-1-seronegative IVDUs were analyzed for HIV-1 infection. HIV-1 nucleic acid was detected only after the use of polymerase chain reaction (PCR) in three of eight CNS samples from HIV-seropositive IVDUs but not in samples from seronegative subjects. In situ hybridization confirmed that HIV-1 DNA sequences were in cells in the CNS parenchyma of two of the three positive samples. This study demonstrates that HIV-1 can infect human fetal CNS tissue in vivo, but that the use of PCR may be necessary for its detection.

Early Myelination in the Human Fetal Lumbosacral Spinal Cord: Characterization by Light and Electron Microscopy

ABSTRACT Myelination in the human central nervous system is well documented after 20 weeks of gestation (WOG). However, earlier stages of this process have not been described in detail, although it is assumed that human myelinogenesis is similar to that observed in other animals. We used light and electron microscopy to study myelination in the human lumbosacral spinal cord during the second trimester of gestation. The kinetics of myelin-associated gene expression were analyzed by immunocytochemistry using antibodies to the myelin markers myelin basic protein (MBP) and 2‘,3’-cyclic nucleotide 3‘-phosphodiesterase (CNPase). These studies show that in 12–13 WOG specimens, occasional MBP-positive processes are found in developing white matter in areas distinct from the root entry zones. At this time, ultrastructural study revealed early investment of axons by glial processes and rare compacted myelin. CNPase staining was qualitatively and quantitatively less than that of MBP. The numbers of MBP- and CNPase-positive myelin sheaths increased with time, and by 24 WOG many were evident in all areas of the spinal cord except in the corticospinal tracts. Ultrastructural study of corresponding areas revealed many thin lamellae of compact myelin. This study provides initial normative data for early human myelination in the lumbosacral spinal cord and may serve as a baseline for future developmental and pathological studies.

Placenta accreta encountered during dilation and evacuation in the second trimester

Objective To assess the frequency of placenta accreta encountered during dilation and evacuation (D&E) in the second trimester. Methods Among 16,827 second-trimester D&E procedures performed at our hospitals and clinics, seven cases of placenta accreta, either suspected clinically or proven histologically, were encountered. These cases were analyzed for history of prior cesarean delivery, placenta localization, and histology of hysterectomy specimens. Results Six of the seven cases suspected clinically were confirmed histologically. All placenta accreta patients had at least one cesarean delivery (mean 1.7), and five had a preoperative sonogram demonstrating some form of placenta previa. The prevalence of clinical placenta accreta encountered during D&Es in the second trimester was 0.04%, the same as that reported for placenta accreta diagnosed clinically in the third trimester. Conclusion Placenta can be a potential complicating factor in the patient undergoing D&E in the second trimester.

Temporal and Spatial Expression of Major Myelin Proteins in the Human Fetal Spinal Cord during the Second Trimester

Irhmunohistochemical identification of myelin basic protein (MBP) is a sensitive method for assessing myelination in the human fetal central nervous system (CNS). However, the temporospatial relationship of expression of two other major myelin proteins, proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) to that of MBP during fetal development has not been assessed in human tissues. Vibratome sections of cervical, thoracic and lumbosacral levels from 37 normal spinal cords of ≤10 to 24 gestational week (GW) fetuses were analyzed using immunohistochemical methods. Using light microscopy, MBP was the first oligodendrocyte marker detected, present by 10 GW at more rostral levels. PLP and MAG were detected rostrally between 12 to 14 GW. All myelin proteins were expressed in anterior to posterior and rostral to caudal gradients. By the late second trimester, expression of MBP, PLP and MAG was noted in all locations in the spinal white matter except for the corticospinal tract. Expression of MAG was particularly marked in the posterior root entry zone and propriospinal tracts. The results suggest that PLP and MAG are expressed later than MBP but follow similar spatial gradients.

Neuroanatomical localization of myelin basic protein in the late first and early second trimester human foetal spinal cord and brainstem.

SummaryThe temporal and spatial expression of myelin basic protein in the first and second trimester human foetal spinal cord and brainstem from 9 to 20 gestational weeks was determined by immunocytochemistry in sections of cervical, thoracic and lumbosacral levels from 41 human foetal spinal cords and ten brainstems. Myelin basic protein-positive oligodendrocytes were observed peripheral to the ependyma at 9–10 gestational weeks. Oligodendrocytes expressing myelin basic protein were seen at 10–12 gestational weeks in the anterior and lateral funiculi. Myelin basic protein was detected later in the posterior funiculi than in the anterolateral white matter and most spinal cord tracts could not be identified by means of variation in myelin basic protein expression. Mylein basic protein was found in the midline of the brainstem at ten gestational weeks and spread laterally during the second trimester. We conclude that in the human foetal spinal cord, myelin basic protein is present by 10 gestational weeks in the anterolateral cervical spinal cord and midline of the brainstem. It is expressed in a rostral-to-caudal and anterolateral-to-posterior manner in most tracts of the spinal cord. However, an exception to these findings is that the fasciculus gracilis, upon developing into a defined region, had more myelin basic protein-positive cells at the lumbar level than in more rostral regions. Definition of the kinetics of myelin basic protein expression in the normal human foetal spinal cord provides a baseline for study of aberrant myelination and demyelination.

Localization of microglia in the human fetal cervical spinal cord.

Differential morphologic subtypes of microglia have been identified in the human fetal frontal cerebrum using a lectin, Ricinus communis agglutinin 1 (RCA-1), and a monoclonal antibody, EBM-11. In this report, microglia were characterized in the human fetal cervical spinal cord. RCA-1-positive microglia were ramified in the developing gray matter while in the developing white matter they had a less differentiated (ameboid) appearance. EBM-11, a monoclonal antibody that recognizes CD68 on human macrophages, and microglia labeled only ameboid-type microglia in the developing white matter. This suggests that distinct subpopulations of microglia exist, which may represent different stages in microglial development, and that CD68 may be a differentiation marker for less mature forms. Therefore, cytologically less differentiated forms of microglia appear to be associated with myelination.

mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure.

Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric α-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 × 106 cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17β-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues.

Immunophenotypic characterization of human fetal liver hematopoietic stem cells during the midtrimester of gestation

OBJECTIVE Our purpose was to define the extent to which gestational age influences the number of fetal liver cells that coexpress phenotypic markers associated with hematopoietic stem cells and major histocompatibility antigens. STUDY DESIGN Fetal liver cells from abortuses of 9 to 24 weeks of gestation were studied (n = 61). Low-density nucleated liver cells were isolated on a discontinuous density gradient and subsequently incubated with antibodies that recognize markers of hematopoietic stem cells (i.e., CD33, CD34, CDw90, CD117, and CD123). Human leukocyte antigen class I (A, B, C) and class II (DR) antigens were also determined on these cells. Each sample was analyzed by immunocytochemistry and flow cytometry. Analysis of variance was used for statistical analysis. RESULTS Of the markers measured, only the percentage of CD123-positive cells increased significantly with gestational age (p < 0.01). The percentage of triple-positive cells (CD34+, CD117+, and CD123+) increased with age but did not reach significance (p = 0.05). Human leukocyte A, B, and C antigens were expressed on all nucleated cells from 9 to 24 weeks of gestation. Human leukocyte DR antigen, however, was expressed only on 50% of these cells. The percentage of cells that expressed both hematopoietic stem cell markers and DR antigen did not vary with gestational age. CONCLUSIONS From 9 to 24 weeks of gestation the number of human fetal liver hematopoietic stem cells that coexpress major histocompatibility antigens increases with advancing gestational age, largely because the percentage of these cells remains constant while the liver mass increases.