Circulating cell-free fetal DNA for the detection of RHD status and sex using reflex fetal identifiers.

OBJECTIVE: To determine the sensitivity and specificity of circulating cell-free fetal DNA in determining the fetal RHD status and fetal sex. METHODS: Maternal blood was collected in each trimester of pregnancy from RhD negative nonalloimmunized women. Whole blood was centrifuged, separated into plasma and buffy coat, and frozen at -80°C. DNA analysis was conducted via allele-specific primer extensions for exons 4, 5, and 7 of the RHD gene and for a 37-base pair insertion in exon 4 (RHD pseudogene; psi) three Y-chromosome sequences (SRY, DBY, and TTY2), and an extraction control (TGIFL-like X/Y). RhD serotyping on cord blood and gender assessment of the newborns were entered into a Web-based database. RESULTS: One hundred twenty women were enrolled. The median gestational age at the first venipuncture was 12.4 (range: 10.6-13.9) weeks with 120 samples drawn; 118 samples were drawn at 17.6 (16-20.9) weeks; and 113 samples at 28.7 (27.9-33.9) weeks. Overall accuracy for RHD was 99.1%, 99.1%, and 98.1% for each trimester and was 99.1%, 99.1%, and 100% for fetal sex determination. CONCLUSIONS: Fetal RHD genotyping and sex can be very accurately determined in all three trimesters using circulating cell-free fetal DNA in the maternal circulation.

Isolation and characterization of novel presenilin binding protein.

Approximately 50% of familial Alzheimer's disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.

Myeloperoxidase polymorphism is associated with gender specific risk for Alzheimer's disease.

Myeloperoxidase (MPO) is a myeloid-specific enzyme that generates hypochlorous acid and other reactive oxygen species. MPO is present at high levels in circulating neutrophils and monocytes but is not detectable in microglia, brain-specific macrophages, in normal brain tissue. However, an earlier study indicated that MPO is present in macrophage-microglia at multiple sclerosis lesions, suggesting that reactivation of MPO gene expression may play a role in neurodegenerative diseases involving macrophage-microglia. In the present study, MPO is shown to colocalize with amyloid beta (Abeta) in senile plaques in cerebral cortex sections from Alzheimer's disease (AD) brain tissue. Microglia costaining for MPO and CD68 are closely associated with plaques, suggesting that plaque components induce MPO expression in microglia. In support of this interpretation, treatment of rodent microglia with aggregated Abeta(1-42) was shown to induce MPO mRNA expression. Also, the ApoE4 allele, the major AD risk factor associated with increased Abeta deposition, was shown to correlate with increased MPO deposition in plaques (P = 0.01, ANOVA). Finally, a genetic polymorphism links MPO expression to Alzheimer's risk, in that a higher expressing SpSp MPO genotype was associated with increased incidence of AD in females, and decreased incidence in males (P = 0.006). These findings suggest that the MPO polymorphism is a gender-specific risk factor for Alzheimer's disease.

Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis.

The myeloperoxidase enzyme (MPO) is expressed specifically in myeloid cells and catalyzes the formation of hypochlorous acid and other cytotoxic oxidants. We previously reported that two alleles of MPO exist which differ in promoter strength due to a base difference in an Alu-encoded hormone response element. The present study shows that the higher expressing MPO genotype is overrepresented in early onset multiple sclerosis in females, implicating MPO in this demyelinating disease. Contrary to the general conception that macrophages lack MPO, immunohistochemical analysis shows that MPO is present in microglia/macrophages in and around MS lesions as shown by colocalization with major histocompatibility antigens HLA-DR and phagocytized myelin. Also, MPO mRNA sequences are detected in cDNA derived from isolated human adult microglia. This is the first evidence that MPO is present in microglia/macrophages at MS lesions, that MPO gene expression occurs in microglia and that MPO plays a role in MS pathogenesis as shown by the allelic disequilibrium in early onset disease.

Expression of the neuron-specific glycoprotein GP50 by granule cell cultures.

The expression and properties of the neuron-specific glycoprotein, GP50, by neonatal rat granule cells grown in primary tissue culture were studied using the monoclonal antibody MabSM-GP50. GP50 was expressed by granule cells in culture but not by astrocytes or PC12 cells that had been induced to differentiate with nerve growth factor. Immunocytochemical staining of granule cell cultures demonstrated that immunoreactivity was concentrated in the cell body. Although the amount of GP50 increased slowly throughout the culture period the maximum level of expression in vitro was markedly lower than that observed in cerebellum of the comparable age. Radiolabelling of cell surface proteins by lactoperoxidase-catalyzed iodination demonstrated that GP50 was expressed on the surface of granule cells. Following phase-partitioning of granule cells in the presence of Triton X-114, 75% of GP50 was found to be present in the detergent phase, indicating that it is an integral membrane protein. Sucrose density gradient centrifugation of Triton X-100 extracts of granule cells resolved GP50 into two components with sedimentation coefficients of 3.6S and 7.3S. The 3.6S species (Mr 42,000 Da) accounted for greater than 80% of the total, indicating that GP50 is present predominantly in a monomeric form within the membrane. These properties are similar to those of GP50 expressed in P12 cerebellum but contrast with the behavior of GP50 from mature brain, in which the 7.3S, hydrophilic form is the predominant species. The results suggest that the mature expression of GP50 may be dependent upon the histotypic pattern of development which occurs in vivo.

Postnatal development of a granule cell-enriched, neurone-specific glycoprotein, gp50, in normal and thyroid-deficient rats.

Glycoprotein gp50 is a neurone-specific, granule cell-enriched glycoprotein that is also a major component of isolated synaptic membranes. Here, we describe the use of a monoclonal antibody, mab SM gp50, to study the postnatal development of gp50 in the brain of normal and thyroid-deficient rats. Radioimmunoassay, enzyme-linked immunosorbent assay, and Western blotting show that gp50 is not detectable in brain until postnatal day 4 (P4) in both forebrain and cerebellum. In forebrain, the rate of increase of gp50 levels is maximal between P12 and P20. It is somewhat later in cerebellum, where peak levels are attained between P30 and P35. Immunocytochemical studies show little detectable gp50-like immunoreactivity before P16, and the staining is still weak, relative to adult tissue, at P25. The intense staining of the granule cell layer characteristic of adult cerebellum predominantly appears after P25. Development of gp50 is severely retarded in the cerebellum of thyroid-deficient rats, particularly during the second and third postnatal weeks. However, by the fourth postnatal week, gp50 levels in normal and hypothyroid animals are comparable. The results indicate that significant alterations in the pattern of gp50 expression continue to occur at a late stage of cerebellar development. In particular, the increase in immunocytochemical staining of the granule cells after P25 is striking in that by this time most major events associated with cerebellar development are essentially complete.

Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC.

We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.

Molecular characterization of GP50: a neuron-specific, synaptic-enriched glycoprotein.

The molecular properties of the neuron-specific, synaptic-enriched glycoprotein GP50 have been investigated with the aid of the monoclonal antibody MabSM-GP50. GP50 immunoreactivity was detected in the brains of the frog, trout, pigeon, snake, rabbit, mouse, cow, and human, although variation in quantity and electrophoretic mobility of the immunoreactive protein between species was apparent. Deglycosylation of synaptic membranes (SMs) with endoglycosidase H, peptide:N-glycosidase F, trifluoromethane-sulfonic acid, and alkaline sodium borohydride indicated that GP50 is associated primarily, if not exclusively, with high-mannose and/or hybrid-type oligosaccharides and lacks complex N-linked and O-linked sugar chains. GP50 remained associated with the membrane fraction following extraction of SMs with alkaline sodium carbonate, was partially (55%) present in the detergent phase following the phase partitioning of SMs in the presence of Triton X-114, and was resistant to proteolytic digestion with trypsin when present as a component of intact membranes. Taken together, these results indicate that GP50 is an integral component of the SM. Sucrose gradient centrifugation of Triton X-100 extracts of SMs or of forebrain and cerebellar homogenates resolved GP50 into two fractions with sedimentation coefficients of 3.6S and 7.3S, which accounted for 45 and 55% of the total, respectively. The 7.3S form occurred exclusively in the aqueous phase following partitioning with Triton X-114, whereas the 3.6S species was found in both the aqueous and detergent phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptophysin expression during synaptogenesis in the rat cerebellar cortex.

In order to study the mechanisms of synaptogenesis in the rat cerebellar cortex, a library of monoclonal antibodies has been generated against proteins of the isolated synapse. One recognizes a glycosylated 38 kDa protein that is concentrated in the synaptic vesicle fraction and resembles synaptophysin biochemically in its molecular weight, charge, and pattern of glycosylation. In the adult cerebellar cortex, the antisynaptophysin(mabQ155) immunoreactivity is codistributed with synapses. Immunoreactivity is strongest in the molecular layer where punctate deposits of reaction product outline the Purkinje cell dendrites. Discrete small profiles, consistent with the distribution of basket cell axon terminals, surround the Purkinje cells, and in the granular layer the synaptic glomeruli are intensely stained. There is no immunoreactivity in the white matter axon tracts. Electron microscope immunocytochemistry confirms the synaptic location of the antigen and suggests that the reaction product is associated with synaptic vesicles. Both round and flat vesicle populations are immunoreactive. Antisynaptophysin(mabQ155) has been used to follow synaptogenesis in the developing rat cerebellum. In the newborn rat (P0), despite the paucity of synapses, there is some specific immunoreactivity, especially in the subcortical white matter. Electron microscopy shows that the antigenicity is associated with vesicles within growth cones, filopodia, and immature axon profiles. During development, antisynaptophysin immunoreactivity increases progressively, along with the maturing cell populations, for both the granule cell-Purkinje cell and the mossy fiber-granule cell synapses. Quantitative biochemical analysis confirms the cytochemical results. These data suggest that neuronal growth cones express a synapse-specific antigen before complete morphological synapses are present.

Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody.

Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-beta-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue.

Multifocal adenoid cystic carcinoma of the lip.

A 62-year-old Caucasian woman had several distinct and separate adenoid cystic carcinomas which involved both sides of the upper lip. Microscopic examination of the surgical specimens suggested that at least one of these malignant neoplasms arose from a pre-existing basal-cell adenoma.

Malignant hyperthermia during general anesthesia: medicolegal considerations.

Malignant hyperthermia is a pathophysiologic process, the occurrence of which is nearly impossible to predict, the diagnosis difficult to discover rapidly, and the treatment unsuccessful in the majority of cases as is borne by the high mortality rate. It is in almost all instances, a fortuitous event, and liability will most probably not be imposed unless the plaintiff can establish by way of expert testimony that defendant-anesthesiologist departed from acceptable methods of care. Definite departures from the present standard of care could be: (1) administering a potent general anesthetic utilizing halogenated inhalation agents and depolarizing muscle relaxants to an individual who has undergone a previous malignant hyperthermia episode, and possibly to a member of his immediate family; (2) failing to have available appropriate resuscitative equipment; and of course, (3) lack of diligence and due care in attempting to treat a case of malignant hyperthermia. It seems, at this point, that serum CPK levels and constant temperature monitoring are additional safeguards, but are not part of the standard of care imposed upon the anesthesiologist.