Pathology-important advances in clinical medicine: assessing fetal lung maturity

The Scientific Board of the California Medical Association presents the following inventory of items of progress in pathology. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, research workers or scholars to stay abreast of these items of progress in pathology that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Pathology of the California Medical Association and the summaries were prepared under its direction.     

Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

Medical geneticists confront ethical dilemmas: cross-cultural comparisons among 18 nations.

To provide a basis for international discussion of ethical problems, we studied responses of medical geneticists in 18 countries to questionnaires about 14 clinical cases and five screening situations. Of 1,053 asked to participate, 677 (64%) responded. There was greater than or equal to 75% consensus on five cases involving (1) disclosure of (1) conflicting diagnostic findings, (2) disclosure of ambiguous results, (3) disclosure of controversial interpretations, (4) protection of mother's confidentiality in cases of false paternity, and (5) nondirective counseling about 45,X and XYY syndrome. A majority (51%-60%) would disclose the diagnosis to relatives at risk for Huntington disease or hemophilia A, against the patient's wishes; would disclose which parent carries a translocation causing Down syndrome; and would disclose XY genotype in a female. As reproductive options for patients with disorders not diagnosable prenatally, 84% would discuss artificial insemination by a donor, 66% would discuss in vitro fertilization with donor egg, and 46% would discuss surrogate motherhood. In all, 85% would perform prenatal diagnosis for (or would refer) parents who refuse abortion, 75% for maternal anxiety, and 42% for selection of fetal sex. Screening questions showed that 72% believed that workplace screening should be voluntary and that results should be confidential.     

Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.

We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components.     

Antibody repertoire development in fetal and neonatal pigs. VII. Characterization of the preimmune kappa light chain repertoire

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vkappa genes in the porcine preimmune repertoire belong to three subfamilies of Vkappa genes that share 87% sequence similarity with human IGKV2. This porcine Vkappa family also shares sequence similarity with some, but not all, Vkappa genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess approximately 60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jkappa segments, complete with recombination signal sequences and separated by approximately 300 nt, were identified approximately 3 kb upstream of a single Ckappa. Surprisingly, Jkappa2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use approximately 10 IGVK2 genes from three of six subfamilies and preferentially one Jkappa segment to generate their preimmune kappa repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.     

Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells.

Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.     

Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice.

The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described. Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter. In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region. In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus. Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein. Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis. Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus. The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease.     

Lipids of chicken epidermis

The lipids from chicken epidermis were analyzed by a combination of quantitative thin-layer and gas-liquid chromatography and by chemical and spectroscopic methods. The lipid groups present included wax diesters (34%), triglycerides (32%), sterols (11%), phospholipids (11%), nonphosphorus-containing sphingolipids (3%), beta-D-glucosylsterols (3%), 6-O-acyl-beta-D-glucosylsterols (2%), steryl esters (1%), cholesteryl sulfate (1%), and free fatty acids (1%). The major phospholipids were phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, and the sphingolipids included ceramides, glucosylceramides, O-acylceramides, and O-acylglucosylceramides. Glucosylsterols and acylglucosylsterols have not been found in mammalian skin, and may be relevant to the evolutionary history of the epidermal water barrier. The wax diesters contained mainly 16-, 18-, and 20-carbon saturated fatty acids esterified to 20- through 24-carbon threo and erythro 2,3-diols, while the chicken epidermal triglycerides contained some very long-chain (26-40 carbon) saturated fatty acids. These wax diesters and unusual triglycerides may be of significance in human health.     

The 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd-restricted cytotoxic T lymphocytes from mice primed by infection.

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.