Rubella cataract in vitro: Sensitive period of the developing human lens.

The clinically known sensitive period of rubella cataract was studied in vitro by infecting 79 human eye rudiments from embryos aged 4-10 wk with rubella virus. The course of the infection was followed by histological and indirect immunofluorescence methods. Of the rudiments, 12 pairs were in the lens placode or open-lens-vesicle stage, 40 already had closed lens vesicles and in another 27 closed-stage pairs an incision was made in the lens capsule before infection to allow the virus to enter the lens. Uninfected controls differentiated well in vitro for 4-6 wk. The eye rudiments infected in the open-lens-vesicle stage showed lens fiber destruction and viral antigens within the lens. No damage or viral antigens were detected in rudiments infected in the closed stage unless the lens capsule was incisedmwhen this was done, however, fiber damage ensued and viral antigens appeared. The lens capsule was concluded to form a protective barrier around the sensirive fibers at the time of closure of the lens vesicle, confirming the earlier hypothesis and clinical findings.

Shift in collagen type as an early response to induction of the metanephric mesenchyme.

Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.

Drug-induced teratogenesis in vitro: inhibition of calcification by different tetracyclines.

Inhibition of calcification in embryonic bone rudiments was studied in the presence of several tetracyclines at three different concentrations. Different criteria for calcification and different concentrations of tetracyclines yielded parallel results and showed significant differences in the inhibitory action of the various compounds. The clear-cut results indicate that the test-system that was developed may be useful for the comparison of various teratogens under simplified controllable conditions.

Morphogenetic interaction of presumptive neural and mesodermal cells mixed in different ratios.

Cells of the presumptive forebrain region and axial mesoderm of Triturus neurulae were disaggregated and combined in different ratios. The differentiation of the central nervous systen in these explants was dependent on the relative amount of mesodermal cells present: an increase of mesodermal cells resulted in a corresponding increase in the frequency with which caudal structures of the central nervous system developed and a gradual loss of the forebrain formations.

Activin disrupts epithelial branching morphogenesis in developing glandular organs of the mouse.

We report that activin profoundly alters epithelial branching morphogenesis of embryonic mouse salivary gland, pancreas and kidney rudiments in culture, indicating that it may play a role as a morphogen during mammalian organogenesis. In developing pancreas and salivary gland rudiments, activin causes severe disruption of normal lobulation patterns of the epithelium whereas follistatin, an activin-binding protein, counteracts the effect of activin. In the kidney, activin delays branching of the ureter bud and reduces the number of secondary branches. TGF-beta induces a pattern of aberrant branching in the ureter bud derived epithelium distinct from that seen for activin. Reverse-transcriptase polymerase chain reaction, Northern hybridization and in situ hybridization analyses indicate that these developing tissues express the mRNA transcripts for activin subunits, follistatin or activin receptors. Our results are suggestive of a potential role for the activin-follistatin system as an intrinsic regulator of epithelial branching morphogenesis during mammalian organogenesis.

Spemann's heritage in Finnish developmental biology.

The Finnish school of developmental biology can be considered a direct descendant of Spemann's school as both the original technology and the fundamental problems were introduced into Finland by Gunnar Ekman (1883-1937) who had worked for extended periods in Germany. After his early death, the work was continued by Sulo Toivonen (1909-1995), and until 1968 the group explored the mechanisms of primary induction and the subsequent segregation of the central nervous system. The extensive investigations led to the formulation of the "double-gradient" hypothesis and ultimately to its experimental vindication.

Neuronal characteristics in embryonic renal stroma.

The metanephric mesenchyme is considered a homogeneous population of predetermined, but pluripotent cells with a nephrogenic bias. By an inductive stimulus, the mesenchyme is programmed to differentiate into the various epithelial phenotypes of the secretory nephron. A fraction of the mesenchymal cells, however, remains in the interstitium between the nephrons and differentiates into spindle-shaped, clear-cytoplasmic renal stroma. We have analyzed the molecular nature of these cells in order to discover the specific cell types that could be involved in the morphogenetic processes during kidney differentiation. In situ hybridization reveals neurofilament light protein mRNA, and immunohistology shows neurofilament light and medium proteins in the stromal cells around kidney tubules. By immunohistochemistry these peritubular stromal cells can be distinguished from the neuronal cells of the renal microganglion: the peritubular stromal cells are neurofilament-positive but L1 neural cell adhesion protein-negative, whereas the neuronal cells with axonal extension are both neurofilament-positive and L1 neural cell adhesion protein-positive. Proliferation index of the stromal cells was low as compared to tubular cells, as shown by bromodeoxyuridine incorporation.

Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates.

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.

[3H]-adenine metabolism and radiation damage during in vitro development of the kidney.

We followed the early post-induction changes in nucleic acid synthesis of the metanephric kidney anlage in vitro. Enhanced incorporation of [3H]-thymidine and [3H]-adenine was detected, but several factors were shown to influence the interpretation of such in vitro experiments. The incorporation is dependent not only on the stage of development of the target organ but also on its transfer to organ culture, as early rudiments require an "adaptive" pre-cultivation to stabilize their metabolism; at more advanced stages growth and DNA synthesis proceed without delay. Another potential artifact is radiation damage which is caused by the incorporated radioisotope and can be detected in prolonged cultures. A [3H]-adenine pulse of more than 1 microCi/ml for 2 hr leads to definite growth retardation, and a 10-microCi/ml pulse causes extensive cell death and atrophy on a 4- to 6-day subculture. The radiation damage is dose-dependent and of variable severity in the different cell lineages within the organ. Since the radioisotope doses were in the range of those commonly used for monitoring cell proliferation and metabolism, we stress the risk of obtaining artifactual results, especially in prolonged cultures after pulse-labeling.

Infections and other maternal factors as risk indicators for congenital malformations: a case-control study with paired serum samples.

An obstetric population of 48,000 individuals was prospectively followed up for evidence of possible teratogenic factors that might be associated with congenital malformations. Serum samples from 274 mothers of defective children and from paired controls were collected during early pregnancy and approximately one month after delivery and tested for antibodies against ten different viruses, Mycoplasma pneumoniae, and Toxoplasma. These data were supplemented with clinical information on infections, other diseases, drug intake, and other potentially teratogenic factors during pregnancy. Mothers of defective children had more seroconversions (fourfold or greater increase in titer) than the controls, 123 vs. 86. This difference was mainly due to an increase in herpes simplex viruses 1 and 2, cytomegalovirus, varicella-zoster virus, and Toxoplasma titers. In addition, the number of reported diseases during the pregnancy, the intake of drugs (especially analgesics and hormones), and the number of earlier abortions were greater in the mothers of the defective children than in the controls.

Defects of the central nervous system in Finland: V. Multivariate analysis of risk indicators.

Based on the Finnish Register of Congenital Malformations a search was undertaken for associations between defects of the central nervous system [CNS] and various selected risk indicators. The study material consisted of 710 cases of CNS defects and their time-area matched pair controls. 259 cases of polydactyly and their controls were also compared with a view of demonstrating the possible biases introduced by the case-control method. The present report is a multivariate analysis of previously observed associations, and the linear logistic regression model was used for the elimination of confounding factors. Risk indicators remaining after the analysis were high maternal age, multipara with previous stillbirths and defective children, especially defects of the CNS. Maternal influenza, especially in combination with the intake of salicylates, was also associated with an increased risk.

Incidence and secular trends of congenital limb defects in Finland.

The total incidence rate of three selected congenital limb defects was 12.8 per 10 000 births in Finland from 1964 to 1977; 1235 cases were reported to the Registry of Congenital Malformations. The incidence rate of reduction limb deformities was 5.0 per 10 000 births (481 cases), of polydactyly 5.8 (559) and of syndactyly 3.1 (299), respectively. The total incidence rate and the rates for all the subgroups had a statistically significant increasing trend even after the incidence rates were compared to the reporting rate of all malformations (p less than 0.01). No significant variations in seasonal distributions were found. In 69% of the reduction deformities only one or both upper limbs were affected, 20% of the children had only lower limb defects, and 11% of the cases had defects in both upper and lower limbs. In 28% of the cases additional malformations were reported. For comparison, incidence rates of the selected limb defects from 13 other national surveillance systems are presented.

Risk indicators of reduction limb defects.

The birth of a child with a reduction limb defect (RLD) was evaluated in relation to vaginal bleeding, threatened abortion, and other complications of pregnancy, placental weight, birth weight, family history, parental age, and the outcome of previous pregnancies. The material consisted of 453 cases of reduction limb defect and an equal number of non-malformed controls matched for time and place. The children were born in Finland during 1964-77. The cases with reduction limb defect without additional malformations were analysed separately. Statistically significant associations were found between the occurrence of reduction limb defect and the following risk indicators: vaginal bleeding, threatened abortion, duration of gestation under 37 weeks, placental weight 400 g or less, birth weight 2500 g or less, and any type of malformation in the relatives. Vaginal bleeding indicated the risk of reduction limb defect to be increased about fourfold; short gestation indicated about twofold risk of reduction limb defect as an isolated malformation. Both low placental weight and low birth weight were associated to a threefold risk of an isolated reduction limb defect. These factors of an abnormal pregnancy indicated even higher risk of reduction limb defect with additional malformations. Preliminary genetic analysis suggests that hereditary factors play no major part in the aetiology of reduction limb defects.

The parotid gland is the main source of human salivary epidermal growth factor.

To clarify the production of human epidermal growth factor (EGF) by different salivary glands, we measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, we studied the presence of EGF in PG and SMG by immunohistochemistry. The mean (geometric) concentrations of EGF in PG saliva (2704 pg/ml, +/- SEM interval 2393-3056 pg/ml, n = 20) was higher (p less than 0.001) than in whole saliva (864 pg/ml, +/- 733-1019 pg/ml, n = 29), which in turn was higher (p less than 0.001) than in mixed SMG + SLG saliva (357 pg/ml, +/- 296-430 pg/ml, n = 16). No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, but only in PG there were prominent EGF deposits in luminal spaces. Our data suggest that EGF is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EGF is mainly from SMG. In mouse almost all salivary EGF comes from SMG and its amount is androgen dependent. Thus there are great differences in sources and regulation of salivary EGF between man and mouse.

Pregnancy outcome in Finland after the Chernobyl accident.

The explosion at the Chernobyl nuclear power plant caused radioactive fallout in Finland in April-May 1986. The fallout was unevenly distributed geographically, and, accordingly, the country was divided into 3 fallout zones. Whole-body radioactivity measurements of randomly chosen persons showed that the regional differences prevailed throughout the following 2 years. Data for legal abortions, registered congenital malformations as well as preterm births and stillbirths of malformed children were collected. The corresponding expected figures were obtained from statistics from 1984 and 1985. No differences in the expected/observed rates of the above parameters were detected.

PIP: The effects of the 1986 Chernobyl accident on Finland are reported. Legal abortions, registered congenital malformations, preterm births, and stillbirths showed no differences in observed vs. expected rates after the accident. The limitations are that the population base is small, the detection system is incomplete, no reliable data are available on early abortions, and germinal mutations were not presently analyzed. Measurement of radiocesium (Cs 134 and 137) was accomplished by the Research Institute for Social Security with a stratified random sample of 380 people. 5 fallout zones differentiated groups, which were then collapsed into 3 groups. Regional differences in levels persisted into 1988. The Finnish Register of Congenital Malformations provided the compulsory data on congenital malformations. The 3 groups (children born before the accident in 1984-85, in the latter half of 1986, and in 1987) were analyzed in the 3 zones during 2 study periods (August-December 1986 and January-December 1987). Monthly analysis of induced abortions showed no increase following the accident. There was a slight decrease in births between January and March 1987, which is believed to be related to anxiety reinforced by public advice. There were no differences between expected and observed congenital defects in the 2 study periods. Preterm and stillbirths followed a similar pattern. The analysis showed no association between the temporal and spatial variations in radioactivity and variable incidence of congenital malformations.

Beta-carotene concentration in buccal mucosal cells with and without dysplastic oral leukoplakia after long-term beta-carotene supplementation in male smokers.

OBJECTIVE: To measure the beta-carotene concentration in buccal mucosal cells in smoking men who had received long-term beta-carotene (BC) supplementation in a controlled trial. To assess the association of cellular BC on the prevalence of dysplasia in oral leukoplakia. DESIGN: An end-of-trial examination of a part of subjects in the Alpha-Tocopherol, Beta Carotene Cancer Prevention Study. SUBJECTS AND METHODS: 343 men who for 5-7 years had received BC (20 mg/d) or alpha-tocopherol (AT) (50 mg/d), or both of these or placebo. BC concentration of buccal mucosal cells was compared in the subjects with BC supplementation (n = 173) to that of those without it (n = 170). Oral mucosae were examined clinically and lesions showing leukoplakia histopathologically. RESULTS: Mean (s.d.) BC concentration in buccal mucosal cells was 7.7 (10.3)mg/kg protein in the subjects who received BC compared to 1.1 (1.7) mg/kg protein in those who did not. The BC concentration in the cells of supplemented subjects correlated with their serum BC levels (P < 0.001). AT supplementation had no effect on BC concentration nor was daily amount of smoking statistically significantly associated with the BC concentration in buccal cells. Altogether 17 subjects showed oral leukoplakia, 7 had dysplasia. In these 7 subjects, the BC concentration in buccal mucosal cells did not differ statistically significantly compared to subjects with only hyperkeratosis (n = 10) (F-test, P = 0.74). CONCLUSIONS: After long-term BC supplementation, BC concentration in oral mucosal cells was 7-fold greater than without supplementation. There was no evidence to support an association between cellular BC concentration and precancerous lesions among the few subjects having them in their oral mucosae.

Sequential cell and tissue interactions governing organogenesis of the kidney.

The complex development of the metanephric kidney illustrates the decisive role of sequential morphogenetic interactions of varying type in organogenesis. In this review the following steps were singled out from this continuous, strictly controlled organogenesis: determination of the mesoderm during the blastula stage, induction of pronephric nephrons during gastrulation, guided migration of the pronephric duct, mesenchyme-controlled branching of the ureter, induced aggregation of the mesenchymal cells of the metanephric blastema, homotypically controlled polarization of the cells in the renal vesicle and remodelling of the vesicle into the S-shaped body, guided migration of endothelial cells into the nephric blastema and the glomerular crevice, and the matrix interaction(s) completing the formation of the glomerular basement membrane with dual origin.

The accident at Chernobyl and trisomy 21 in Finland.

Our objective was to explore whether the radiation fallout in Finland after the accident at the Chernobyl nuclear power plant in April 1986 led to an increased incidence of trisomy 21. In this geographic and temporal cohort study, the country was divided into three zones according to the amounts of radioactive fallout and internal radiation caused by two cesium isotopes. The 518 cytologically verified cases of trisomy 21 were divided into a control group (conceived before the accident), and a study group of children whose expected dates of birth were in the post-accident years 1987-1988, i.e., pregnancies commenced after May 1986. The cases were also divided into three subgroups according to the zones of radiation. There were no significant differences in prevalence of trisomy 21 between the control and study groups nor between the three zones in spite of the significant differences in the levels of radiation and in the body burden that prevailed throughout the study period. Power estimates showed that in the two zones of lower radiation, an increase of 0.5% in the prevalence would have been detected with a power of 0.85, and in the somewhat smaller zone of the highest radiation, with a power of 0.70. The study lends no further support to the view that the low radiation fallout in western Europe would have been causally associated with trisomy 21.

Periodontal bone loss in patients with Down's syndrome: a follow-up study.

The alveolar bone loss in 24 patients with chromosomally verified Down's syndrome, and in a control group of 28 mental retardates from the same institution, was examined using orthopantomography. The same patients had been examined 5 years earlier, and the present results were compared with the former. During this 5-year period the patients did not receive any periodontal treatment, only daily brushing of the teeth. A clear difference between the two groups was evident at both times: the percentage of affected teeth in the study group was 25% in 1975 and 47% in 1980 as against corresponding figures of 1.8% and 6.8% in the control group. The prevalence rate of bone loss of 5 mm or more showed little or no increase in the patients with Down's syndrome (69% to 75%) whereas in the controls the rate of increase was greater (20% to 43%). In individual teeth, i.e. the mandibular first molars, progress was also more rapid in the controls, 28.3%, as compared to 12.7% in the study group.

Recovery of A. actinomycetemcomitans from teeth, tongue, and saliva.

The recovery of actinobacillus actinomycetemcomitans simultaneously from subgingival sites around teeth and dorsum of the tongue and/or saliva was examined in 293 subjects at 444 visits; 295 paired samples were available from subgingival sites and tongue, 171 paired samples from subgingival sites and stimulated saliva, and 137 paired samples from subgingival sites and unstimulated saliva. Sixty-one subjects were periodontally healthy (mean age 20.3 years); 55 exhibited localized juvenile periodontitis (mean age 21.8 years); 176 adult periodontitis (mean age 46.7 years); and 1 prepubertal periodontitis (age 10 years). When A. actinomycetemcomitans was recovered from subgingival sites, it was also found in 56.3%, 69.9%, and 35.9% of the paired samples from tongue, and stimulated and unstimulated saliva, respectively. No difference in the detection rate of A. actinomycetemcomitans from tongue or stimulated saliva was seen between the subjects with healthy or diseased periodontium. When A. actinomycetemcomitans was not recovered from subgingival sites, it was cultured in 6.8%, 2.0%, and 1.4% of the paired samples from tongue, and stimulated and unstimulated saliva, respectively. In search for noninvasive, inexpensive, and easily run sampling methods for the recovery of oral A. actinomycetemcomitans samples from stimulated saliva and tongue may prove useful in clinical periodontology.