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Histochemical study of the differentiation of microglial cells in the developing human cerebral hemispheres.

Applying nucleoside diphosphatase (NDPase) histochemistry, the appearance and differentiation of microglial cells in the developing human cerebral hemispheres were investigated by light and electron microscopy. In the pallium of the 38 days old human embryo, a few round NDPase-positive cells (round cells) were observed in the expanding zone. Although distinct blood vessels had not yet formed within the wall of the pallium, some cellular elements resembling haemopoietic cells were noticed in the expanding zone. In the 51 days old fetus, blood vessels displaying NDPase activity were seen in the mantle and marginal layers, and some invaded the matrix. Several round NDPase-positive cells were distributed, mainly around the vascular sprouts (primitive blood vessels) in the matrix. In the marginal layer, NDPase-positive cells exhibiting short cytoplasmic processes were encountered (poorly ramifying cells). In the 58, 66 and 82 days old fetuses, the round NDPase-positive cells were seen mainly in the matrix or subcortical layer where vascular sprouts were conspicuous and the poorly ramifying cells were in the subcortical and marginal layers. In the two latter fetuses, NDPase-positive cells showing long highly ramifying cytoplasmic processes (highly ramifying cells) were noted mainly in the marginal layer and sometimes in the subcortical layer. In the 5 months old fetuses, numerous NDPase-positive cells were distributed in the mantle, subcortical and marginal layers, and most of them appeared to belong to the populations of the poorly or highly ramifying cells. On the basis of the ultrastructural features, the round cells and highly ramifying cells were regarded as amoeboid cells and microglial cells, respectively. These findings suggest that at least some amoeboid cells are transformed into microglial cells via the stages of poorly ramifying microglial cells, and also that, in the human cerebral hemispheres, appearance of the microglial elements is closely related with vascularisation, especially in the early developmental stages.

Brain

Studies on copper-zinc superoxide dismutase expression in developing human liver and kidney.

CuZn superoxide dismutase levels were found to be high in developing human kidney and liver compared to some other tissues including lung. In kidney, the enzyme was expressed in proximal and distal tubules, loop of Henle and collecting tubules and after 35 weeks of gestation it appeared to be distributed basally in proximal cells and luminally in distal cells. Glomerular structures were generally negative. CuZn superoxide dismutase was widely expressed in developing liver, with hepatocytes and bile duct epithelium demonstrating positivity. The low level of expression of CuZn superoxide dismutase in the glomerulus compared with the tubules was not expected since intrinsic glomerular cells demonstrate greater production of reactive oxygen species in response to some stimuli than do tubular cells. Expression of this enzyme may be determined by the need to generate hydrogen peroxide.

Cytosol

Behavioral research toward prevention of childhood injury. Report of a workshop sponsored by The National Institute of Child Health and Human Development, Sept 3-5, 1986.

At a recent workshop sponsored by the National Institute of Child Health and Human Development, Bethesda, Md, injury and child behavior researchers recommended continued expansion of both directed research on control of specific injuries and basic research on mechanisms that underlie many or all injuries. Prevention of injuries that are not amenable to environmental control will require change in human behavior. Important research questions to address this goal include the following: How do identified risk factors influence the occurrence of injuries? How do children learn behaviors that alter the rate of injury? How do child developmental factors contribute to safety and injury? Innovative methods are needed, such as measure of environmental hazards, exposure to risk, and precursors of serious injuries. These approaches expanded by a broadened base of investigators should help reduce childhood mortality and morbidity due to injuries.

Adolescent

Intelligence at six years in relation to neonatal bilirubin levels: follow-up of the National Institute of Child Health and Human Development Clinical Trial of Phototherapy.

Results of the National Institute of Child Health and Human Development Randomized Controlled Trial of Phototherapy were examined for the relationship of neonatal bilirubin level to neurological and developmental outcome at 6-year follow-up. This analysis focused on 224 control children with birth weight of less than 2000 g. Bilirubin levels were maintained below previously specified levels by the use of exchange transfusion only (24%). Rates of cerebral palsy were not significantly higher for children with elevated maximum bilirubin level than for those whose level remained low. No association was evident between maximum bilirubin level and IQ (Full Scale, Verbal, or Performance) by simple correlation analysis (r = -.087, P = .2 for Full Scale) or by multiple linear regression adjusting for factors that covary with IQ (beta = -.15, P = .58). IQ was not associated with mean bilirubin level, time and duration of exposure to bilirubin, or measures of bilirubin-albumin binding. Thus, over the range of bilirubin levels permitted in this clinical trial, there was no evidence of bilirubin toxicity to the central nervous system. Measures used to control the level of bilirubin in low birth weight neonates appear to prevent effectively the risk of bilirubin-induced neurotoxicity.

Bilirubin

Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone.

Using nondegradative isolation procedures we purified and characterized five major noncollagenous proteins from developing human bone. Small bone proteoglycan I, Mr approximately 350,000 on sodium dodecyl sulfate (SDS), 4-20% gradient polyacrylamide gels has a different amino-terminal sequence of NH2-Asp-Glu-Glu-()-Gly-Ala-Asp-Thr and is not cross-reactive with the small bone proteoglycan II, Mr approximately 200,000 on SDS-gradient polyacrylamide gels. Bone proteoglycan II is 95% N terminally blocked and the small amount that can be sequenced has an amino-terminal sequence (NH2-Asp-Glu-Ala-()-Gly-Ile. . .) that is apparently similar but not identical to a small proteoglycan isolated by Brennan, M.J., Oldberg, A., Pierschbacher, M.D., and Ruoslahti, E. (1984) J. Biol. Chem. 259, 13742-13750 from human fetal placenta membrane. Two bone sialoproteins, each of which migrates at a Mr approximately 80,000 on SDS gels, have also been isolated. Bone sialoprotein I has an amino-terminal sequence of NH2-Ile-Pro-Val-Lys-Gln-Ala. . . which is different from that of bone sialoprotein II with an amino-terminal sequence of NH2-Phe-Ser-Met-Lys-Asn-Leu. . . The two bone sialoproteins do not cross-react on Western blot analysis. Human bone osteonectin contains a large number of cysteines, more than 90% of which appear to be in disulfide bonds. The N-terminal amino acid sequence of human bone osteonectin was nearly identical to bovine bone osteonectin and had many similarities to a protein found in mouse parietal endoderm (Mason, I.J., Taylor, A., Williams, J.G., Sage, H., and Hogan, B.L.M. (1986) EMBO J. 5, 1831-1837.

Amino Acids

Effects of the vestibular system on human development, part 2: Effects of vestibular stimulation on mentally retarded, emotionally disturbed, and learning-disabled individuals.

In Part 1 of this two-part series, studies concerned with the effects of vestibular stimulation on human development and function were reviewed and some implications for therapy were suggested. In Part 2, three categories of dysfunction with possible links to the vestibular system are discussed. Studies in the category of mental retardation evaluate motor development and sensory preference. Possible vestibular associations with emotional disturbance are examined by review of studies concerned with etiology, motor activity, speech, and clinical observations. A brief review of studies concerned with early identification and speech and language factors of learning-disabled children constitutes the third category. Interpretations are drawn and some implications for therapy are made.

Affective Symptoms

Scientist-administrators at the National Institute of Child Health and Human Development as contributors to the scientific enterprise.

At the National Institute of Child Health and Human Development (NICHD), as in other government research supporting agencies, scientist-administrators who are "program staff" work to accomplish their organization's set of research priorities using established mechanisms for supporting research. At the same time, the definition of their work is given to their interpretation, which, in turn, is guided by their understanding of their scientific discipline and their commitment to it. The tension that may arise between the organization-guided role and the science-guided role is more apparent than real because the major responsibility of "program staff" within the Institute is to cultivate a grant portfolio addressing scientific issues relevant to the mission of the Institute and exemplifying the most advanced research concepts and methodologies. When the overlap between the mission of the Institute and the direction of science is small, the push to increase it leads to new and imaginative solutions that benefit both the Institute and the science.

Administrative Personnel

An immunohistochemical study of bronchial cells producing surfactant protein A in the developing human fetal lung.

A study on immunohistochemical localization of pulmonary surfactant protein A (SP-A) in the developing human fetal lung was performed using a monoclonal antibody, PE10, against human SP-A. At 21 weeks of gestation, a few bronchial cells positive to PE10 were observed to be scattered in the main and segmental bronchi. The number of these cells appears to increase until the 32nd week of gestation, and then decrease thereafter, almost disappearing by 39 weeks. On the other hand, alveolar type II cells and Clara cells positive to PE10 began to appear at 29 weeks, increase in number until around 39 weeks, and remain constant throughout adulthood. A few bronchial glandular cells positive to PE10 were still noticed in the fetal lung. This is the first report of the presence of SP-A-containing cells in the fetal lung. This is the first report of the presence of SP-A-containing cells in the human fetal bronchial epithelium (not Clara cells in the terminal bronchiolus), proving the regularity of the sequential distribution of SP-A-containing cells in the bronchoalveolar system during pulmonary development.

Aged

Effects of the vestibular system on human development, Part I. Overview of functions and effects of stimulation.

Studies concerned with the effects of vestibular stimulation on human development and function are reviewed and some implications for therapy are suggested in this paper. Studies are included that relate to changes in the vestibulo-ocular reflex with age, effects of vestibular stimulation on smiling, crying, general activity, and visual attentiveness of infants; and studies that show that typical sequelae to vestibular stimulation are reduced following long-term stimulation. The importance of reviews of the literature and transference of research results to therapeutic use are stressed, with the implication that vestibular-based therapy will improve as occupational therapists become more aware of related research.

Adolescent

Possible sex differences in the developing human fetal brain.

Left-right regional volumetric asymmetries in five telencephalic regions were studied in the developing human fetal brain. Complete series of coronal sections of 21 fetal brains were digitized and regional volumes were integrated. Five regional indices of asymmetry and two overall indices of asymmetry were calculated and compared across the fetal sample. The two most asymmetrical regions in the developing fetal brain were region 1, roughly equivalent to prefrontal cortex, and region 5, which includes striate and extrastriate cortices. Region 5 also manifested a statistically significant sex difference (p less than .02) in the degree of volumetric asymmetry. It appears that striate-extrastriate cortices are far more asymmetrical in male brains than in their female counterparts (M = 33%; F = 13%). Overall indices of asymmetry indicated that, on the average, volumetric asymmetries in the male brain favor the right hemisphere. In contrast, the human fetal female is likely to have two hemispheres of the same size or a left hemisphere that is slightly larger than its right counterpart. We believe that these results support the hypothesis that testosterone in utero may lead to a more rapid growth of the right hemisphere or, alternatively, retard the growth of the left hemisphere.

Brain

[Neuroblast differentiation and migration in the developing human spinal cord in the 1st half of prenatal ontogenesis].

The processes of neuroblasts differentiation and migration in the developing human spinal cord in the first half of antenatal ontogenesis were studied by the Golgi method. The neuron development was traced from the neuroepithelial matrix cells to the maturing neurons. A possible role of a special neuroblast outgrowth as a channel for its nucleus migration to the definite typological position of the mature neuron is suggested.

Cell Differentiation

Distribution and characterization of anionic sites in the basal lamina of developing human amniotic epithelium.

We have studied the distribution of anionic sites in the basal lamina of developing human amniotic epithelium by using the cationic stain ruthenium red. Amnions at 7-12 weeks of gestation and at term contained ruthenium red-positive granules in a quasi-regular array on both the cellular and interstitial sides of the lamina densa. In order to characterize the anionic sites, small pieces of amnion were incubated in the presence or absence of either chondroitinase ABC, neuraminidase, Streptomyces hyaluronidase, or heparitinase in appropriate buffer systems. Incubation in the presence of heparitinase resulted in the complete disappearance of the basal lamina-associated granules, but other enzymes tested had no demonstrable effect on these granules. We conclude that the anionic sites associated with amnion basal lamina, and demonstrable with ruthenium red, consist of glycosaminoglycans rich in heparan sulfate, probably present as heparan sulfate proteoglycan. Because amniotic fluid has a low protein content and amniotic epithelium (at least at term) lacks tight junctions, we postulate that the heparan sulfate proteoglycan associated with the amnion basal lamina may have an important function as a permeability barrier to anionic macromolecules.

Amnion

Myelin-associated glycoprotein in the developing human retina.

The immunohistochemical presence of myelin-associated glycoprotein (MAG) in Müller cells of the developing human retina was examined with rat monoclonal antibodies to MAG and the peroxidase antiperoxidase (PAP) method of Sternberger. Retinas of various developmental stages ranging between 9-31 gestational weeks were stained. There was no staining in the retinas of 9-12-week embryos. Between 13-16 gestational weeks the staining was faint and located mostly in the inner and middle portion of the retina, primarily around the optic nerve head. After midterm, Müller cells invariably stained through all retinal layers. The staining increased gradually up to the twenty-third gestational week, when it reached the level found in the retinas of newborn children.

Antibodies, Monoclonal

Gastric secretory function in the developing human stomach.

Little data exists regarding the activity of gastric parietal cells in the very immature infant. Therefore we have examined the developing human stomach for the presence and location of parietal cells, using both standard histological methods and antibodies to the H+/K+ ATPase (proton pump) and intrinsic factor, in 35 fetuses (ranging from 13-28 weeks) and in five infants (2-21 weeks). Parietal cell activity was noted in the body, antrum and pyloric regions in all the fetal specimens examined. However, this activity was much more limited in the infant specimens. We have noted that from the end of the first trimester parietal cells are present in a mature, functional form with the potential to secrete both gastric acid and intrinsic factor.

Embryonic and Fetal Development

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones.

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.

Bone Development

Localization of the expression of type I, II, III collagen, and aggrecan core protein genes in developing human articular cartilage.

The expression of mRNAs for collagen types I, II, III and for aggrecan core protein was studied in developing human femoral cartilage by in situ hybridization, with special attention given to the cartilage covered by the perichondrium and to the articular surface. In parallel, the synthesis of the related proteins was monitored by immunohistochemistry. The cells metabolically active for type I and type III collagen expression were identified by hybridization using [32P]-labeled cDNA clones coding for human alpha 1(I) and alpha 1(III), respectively. Type II collagen and core protein mRNAs were detected by hybridization with specific [32P]-labeled oligonucleotide probes. In the femoral heads of one 22-week old fetus and of one newborn, our in situ hybridization and immunohistochemical analysis revealed that chondrocytes located immediately subjacent to the perichondrium produced collagen types I, II, III as well as aggrecan; whereas only type II collagen and aggrecan gene expression was detected deeper in the cartilage covered by the perichondrium. This observation supports the hypothesis that the inner cell layers of perichondrium are chondrogenic, with a transient state where cells express all the markers studied here. At the articular surface different patterns of expression were observed at the two developmental stages. After 22 weeks of fetal development only collagen types I and III were expressed by the surface zone cells while in the newborn cartilage, these cells expressed all the molecules studied (collagen types I, II, III and cartilage proteoglycan). At both ages the underlying cartilage cells expressed only the cartilage-specific molecules (type II collagen and aggrecan). Thus a progressive transformation of cartilaginous matrix occurs with time from the deep cartilage up to the surface by addition of new components, i.e. aggrecan and type II collagen. These results supplemented by an immunofluorescence analysis on 20-, 26- and 38-week old fetal femoral heads suggest that expression of collagen and aggrecan in the cartilage covered by the perichondrium and in the cartilage at the articular surface are subject to different regulatory mechanisms during development. Furthermore, the appearance of hybridizable core protein and type II collagen mRNAs at the articular surface, closely followed by the appearance of the proteins for which they code, indicates that core protein and type II collagen expression is regulated primarily at the transcriptional level in this region. Finally, the similar topography observed for the expression of these two proteins suggests that the genes for these two major constituents of cartilage matrix are coordinately regulated during growth of articular cartilage.

Aggrecans

Type IV collagen in developing human lung: a comparison between normal and hypoplastic fetal lungs.

Human lung tissue obtained from 24 aborted fetuses of varying gestational ages (beyond 24 weeks) was examined for immunolocalisation of type IV collagen, using monoclonal antibody. Twelve cases of lung hypoplasia associated with oligohydramnios were compared with 12 normally developed lungs over the same gestational age range acting as control. Type IV collagen was located in alveolar septa and subepithelial basement membranes in all the lungs examined but showed less prominently in hypoplastic lungs than in control lungs of a similar gestational age. The findings suggest a potential role of type IV collagen in maturation of the developing human lung.

Amniotic Fluid

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues.

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.

Adrenal Glands