Inés Wong

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture

The effects of recombinant human growth hormone (GH, 1 µg/ml) and insulin-like growth factor I (IGF-I, 200 ng/ml) on the production of insulin and glucagon by human fetal islet-like cell clusters (ICCs) were studied in tissue culture. ICCs were derived after collagenase digestion and culture of pancreases from 16 fetuses (mean gestational age 15.6 wk). The ICCs were cultured with or without GH or IGF-I for 7 or 31 days. Basal rates of insulin and glucagon production were not altered by GH during the first 17 days of culture, but the release of both hormones was increasingly augmented by GH during the last 2 wk of culture (131% increase in insulin and 85% in glucagon compared with controls). ICCs cultured for 7 days in the presence of GH secreted more insulin when incubated for 120 min in 20 mM than in 2 mM glucose (2.1-fold response, P < .05), whereas ICCs mantained in basal medium did not respond to glucose. GH had no effect on DNA and insulin content or insulin biosynthesis. Exogenous IGF-I caused a 28% suppression of insulin release (P < .05) between days 4 and 10 of culture but induced a 49% increase in the mean secretion rate during the last week (days 25–31, P < .01). Glucagon release was not affected by exogenous IGF-I. In contrast to GH, exogenous IGF-I induced a twofold increase in the DNA content of the 7-day-cultured ICCs. However, insulin biosynthesis and release were markedly suppressed. We conclude that GH influences the functional maturation of human fetal islet cells in vitro. The effects of GH could not be reproduced by the addition of IGF-I, suggesting a direct rather than a somatomedin-mediated action for GH.

A Study of the Structural and Biochemical Development of Human Fetal Islets of Langerhans

The structural and biochemical ontogeny of 14 mid-trimester (12.0–19.5 wk) human fetal endocrine pancreata were examined. Each pancreas was halved at the region of the isthmus into duodenal (D) and splenic (S) portions, and islets, isolated from each of the two portions, were aliquoted and grouped according to diameter into 4 categories: (1) > 300 μm (2) 300–200 μm (3) 200–100 μm (4) 100 μm. At all fetal ages, the number of islets isolated from the splenic half was greater than from the duodenal half, and the number of islets increased as their diameter decreased. The mean total cell number per islet for each of the 4 categories are reported: (1)(D) 30,810 (S) 28,450; (2)(D) 10,970 (S) 10,250; (3)(D) 3,390 (S) 3,460; (4)(D) 1,930 (S) 1,840. Measurement of islet insulin (IRI), glucagon (IRG), and somatostatin (IRS) by radioimmunoassay showed a progressive increase with time in the content of all three hormones in all islets from both halves of the pancreas (with the exception of the smallest islets of <100 μm diameter). After 18 wk, a dramatic increase in the content of all three hormones was observed particularly in the case of the largest (>300 μm) islets. In contrast, pancreatic polypeptide (IRPP) shows no significant increase. With the exception of the smallest islets (100 μm), all splenic-half islets contain a significantly greater content of IRI, IRG, and IRS compared with their duodenal-half match while the content of IRPP is significantly greater in the duodenal-half islets. In addition, all islets with the exception of the largest (>300 μm), contained equal quantities of IRG and IRS and these results were apparent at 12 wk gestation. These data provide new insights into the ontogeny of individual islet cells during the mid-trimesteric period and serve as a basis for future studies of both normal and abnormal pancreatic development.

Effects of infection by HIV-1, cytomegalovirus, and human measles virus on cultured human thymic epithelial cells

A tissue culture system for the growth of human fetal and infantile thymic epithelial (TE) cells has been established and characterized. We have investigated the effects of infection of these cells by human cytomegalovirus (CMV), measles virus, and human immunodeficiency virus type‐1 (HIV‐1). In the case of CMV, morphological changes were apparent by 2–4 days after viral inoculation of infantile TE cells. CMV‐related antigens were detected by immunofluorescence after 12 days, and progeny infectious CMV was recovered from culture media after 18 days. Following infection by measles virus, distinctive, multinucleated giant TE cells appeared in both cultures of fetal and infantile TE cells. Measles virus‐inoculated TE cells displayed an altered phenotype, as revealed by reaction with monoclonal antibodies with specificity for a variety of TE markers. Finally, infection of TE cells by HIV‐1 resulted in cellular disarrangement, increased numbers of Hassalls corpuscles, and multinucleated giant cells. An increase in the number of cells reactive with monoclonal antibodies, specific for Hassalls corpuscles, was observed in the case of cells infected by either measles virus or HIV‐1. These findings suggest that a variety of different viruses can successfully infect thymic epithelial tissue. Because of the important role of the thymus in development of the immune system, it is reasonable to conclude that viral infection of thymic tissue might play an important role in virus‐mediated suppression of immune responsiveness.

Dominantly inherited osteogenesis imperfecta in man: an examination of collagen biosynthesis.

Extract: We have examined control subjects and patients in an effort to discover a metabolic basis for dominantly inherited osteogenesis imperfecta (OI). Studies were carried out in vitro with cultured skin fibroblasts obtained from OI patients, and in vivo on peptide-bound hydroxyproline excretion in urine. Urinary hydroxyproline excretion (milligrams/24 hr) adjusted for age is essentially normal in OI patients, although the mean excretion rate is below average. The latter finding is presumably a reflection of the smaller body mass of OI patients.The OI skin fibroblasts, matched for age of donor, site of biopsy, phase of growth, and generation number in culture, incorporate L-proline into hot trichloroacetic acid (TCA)-soluble protein (collagen) at normal rates. The rate of conversion of proline to hydroxyproline in the nascent polypeptide is also normal in OI. Incorporation of L-lysine was also normal in OI. These findings indicate that peptide synthesis of collagen is not impaired in OI.Rates of galactose incorporation into collagen and the extractability of collagen into normal saline or 0.2 M citric acid were all normal both in OI cells and in the culture medium recovered from the monolayer. These findings, in combination with the urinary data on hydroxyproline excretion in vivo reveal that cross-linking and export of collagen in OI is essentially normal.The elution profile after ion exchange chromatography of fibroblast collagen on carboxymethyl (CM)-Sephadex was also examined. The normal 2/1 ratio of peak 1 (largely α1(I) chains) to peak 2 (largely α2 chains) was found in OI fibroblast extracts, which implies that synthesis and initial aggregation of the two types of polypeptide to yield [α1(I)]2α2 collagen composition is not abnormal in OI.Despite the negative biochemical findings, a consistent defect in the morphology of OI cells was identified in the log phase and the confluent phase of monolayer cultures. The finding is characterized by irregular packing of the aggregated cells and by an irregular tessellated appearance of the individual OI fibroblast. This observation reassures us that the inherited defect is expressed in vitro.Speculation: An abnormality in the primary sequence of polypeptide chain in collagen would be compatible with all of our findings and with the genetics of OI. The mutant allele would affect only about half the products, under the control of only one of the loci determining the polypeptide sequences in collagen chains. Because the OI allele is not expressed in cartilage, a tissue without α2 collagen chains, the defect in OI would perhaps be found in the α2 polypeptide. However, since the α1(II) chain of cartilage differs in amino acid composition and in hydroxylysine-linked carbohydrate from the α1(I) chains of noncartilagenous structures, a defect in α1(I) chains at the nonhomologous residues will also require investigation.

Replication of cytomegalovirus in human thymic epithelial cells

Cytomegalovirus (CMV) has often been cited as a cause of immune suppression in children, yet little is known of the mechanisms through which this agent might affect immune function. We have succeeded in using CMV to productively infect cultured human fetal and infantile thymic epithelial (TE) cells. Morphological changes were apparent by 2–4 days after viral inoculation. CMV-related early antigen (EA) and late antigen (LA) were detected by immunofluorescence after 8 days, and progeny infectious CMV was recovered from culture media after 12–17 days. TE cells that reacted with monoclonal antibodies specific for keratin and for GQ ganglioside were predominant throughout the culture period. In contrast, infection by CMV resulted in a significant decrease in numbers of cells reactive with monoclonal antibodies specific for mesoderm-derived components. Inoculation of TE cells with CMV also caused a diminution in levels of detectable interleukin-1 (IL-1)-related antigen by 17 days after infection.

LACK OF GLUCOSE-INDUCED FUNCTIONAL MATURATION DURING LONG-TERM CULTURE OF HUMAN FETAL ISLET CELLS

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.

246 A STUDY 0F THE STRUCTURAL AND BIOCHEMICAL DEVELOPMENT OF HUMAN FETAL ISLETS OF LANGERHANS

Variably-sized, histologically intact islets were isolated from 14 human fetal pancreases (12.0-19.5 wks. gestation) and grouped according to gestation age, region of the pancreas (splenic and duodenal) and diameter sizes. The mean number of cells/islet, based on DNA/islet was ∼30,000 (>300μm diameter), ∼11,000 (200-300μm), ∼3,500 (100-200μm) and ∼2,000 (<100μm). In both duodenal and splenic islets and all sizes the mean concentration of insulin (IRI) and glucagon (IRG) and somatostatin (IRS), expressed as pg or pg equivalents of insulin, glucagon and somatostatin per islet simultaneously increased significantly in three distinct step-wise developmental tiers: (1) 12.0-14.5 wks, (2) 15.0-18.5 wks, (3) 19.0-19.5 wks. In contrast, pancreatic poly-peptide (IRPP) concentration did not. At all fetal ages and islet sizes, content of IRI, IRG and IRS was significantly greater than IRPP in splenic islets while IRPP was greater in duodenal islets. Mean hormone ratios at all ages and sizes indicate IRI content is significantly greater than IRG and IRPP but not IRS. In summary, a precise pattern of multiphasic hormone development of IRI, IRG and IRS as well as the relative importance of IRS-containing D cells has been uncovered in human endocrine pancreas during fetal ages 12.0-19.5 wks.