Programmed cell death in normal fetal rat lung development

Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.     

Cyclophosphamide inhibits the development of diabetes in the diabetes-prone BB rat

Abstract Aims/hypothesis. Cyclophosphamide has been shown to augment the diabetic process in NOD mouse and BB rat models of Type I (insulin-dependent) diabetes mellitus. Because cyclophosphamide has, however, been shown to increase immunoregulatory cell activity, we examined if cyclophosphamide treatment increases immunoregulatory cell activity and inhibits the diabetic process in BB rats. Methods. The development of insulitis and diabetes was explored in BB rats treated with saline and cyclophosphamide (60 to 175 mg/kg body weight). Subsets of spleen cells were assessed by flow cytometry and cytokine gene expression by RT-PCR. To determine if cyclophosphamide induces immunoregulatory cell activity, the development of diabetes was assessed in BB rats injected with spleen cells from rats treated with saline and cyclophosphamide. Results. All dosages of cyclophosphamide decreased the development of diabetes. The degree of insulitis was lower in pancreata from 55-day-old rats treated with cyclophosphamide than those from controls. Cyclophosphamide caused no alterations in the numbers of NK cells, T-cell subsets, or RT6.1⁺ T cells. The adoptive transfer of spleen cells from cyclophosphamide-treated rats to BB rats inhibited the development of diabetes. Cyclophosphamide treatment decreased IL-12, IL-1β, IL-2, IFN-γ and TNF-α gene expressions in mononuclear spleen cells but IL-4 gene expression increased. Conclusion/interpretation. These findings show that cyclophosphamide treatment decreases the development of diabetes by inhibiting the development of insulitis. This inhibitory action of cyclophosphamide on the diabetic process seems to be mediated by the induction of immunoregulatory cell activity. The suppression of cytokines that promote Th1 cell differentiation by cyclophosphamide treatment could also play a part in the diabetes sparing effect of cyclophosphamide. [Diabetologia (2000) 43: 986–994] Received: 10 December 1999 and in revised form: 13 April 2000     

Elastin metabolism during perinatal lung development in the copper-deficient rat

Metal-chelating drugs were employed to investigate the role of copper (Cu) in elastin metabolism during the period of alveolarization in rat lung. Six groups of pregnant Sprague-Dawley rats were fed one of six semipurified diets, i.e., sufficient or deficient in copper, or the same basal diet containing 0.2% or 0.4% D-penicillamine (DPA), or the same basal diet containing 0.2% or 0.4% triethylenetetramine (TETA). The dams were fed throughout gestation, parturition, and lactation. The pups were then killed postnatally at day 10 and day 21 for the various analyses. At day 21, liver copper in the Cu-deficient pups was 3-5% control levels. In drug-treated groups, liver copper was 16-30% control levels. In the 21-day-old Cu-deficient rats, the concentration of lung elastin was only 75% of its concentration in control. Lung lysyl oxidase activity was lower in Cu-deficient rats, and data for the relative distribution of lung elastin cross-linking amino acids indicated impaired cross-linking in the pups from both the Cu-deficient and the 0.4% DPA groups. Morphologic examination of the lung also indicated dilation of airways in these two groups. Data on the distribution of cross-linking amino acids in elastin samples were also consistent with previous suggestions that impaired cross-linking observed in copper deficiency or from DPA treatment results from different mechanisms. Since the intakes of DPA and TETA were chosen to be in the therapeutic range of intakes used to treat diseases of abnormal copper metabolism, an important feature of these studies is that DPA and perhaps TETA have the potential of impairing normal lung development.     

Regulation of rat pancreatic glucocorticoid receptors by thyroxine during development

The effect of T4 on the development of pancreatic glucocorticoid receptors was studied in normal and adrenalectomized rat pups. Daily injection of T4 (0.1 microgram/g BW) to intact pups starting 3 days before death at 10, 15, and 20 days of age resulted in a precocious increase in pancreatic glucocorticoid-binding capacities. Intact pups made hypothyroid by propylthiouracil feeding exhibited lower glucocorticoid-binding capacities in their pancreata. Scatchard analysis demonstrated an increase in the number of glucocorticoid-binding sites in the pancreata of T4-treated intact rats compared to that in normal intact rats. In hypothyroid groups the number of glucocorticoid-binding sites was much lower than that in normal intact rats. The Kd values, however, were unchanged in hypothyroid, hyperthyroid, and control groups. Rat pups who underwent adrenalectomy at 12 days of age had undetectable plasma corticosterone levels and showed an increase in their pancreatic glucocorticoid-binding capacity 3 days after operation. Replacement of corticosterone resulted in a binding level similar to that in the sham-operated group. However, injection of T4 alone to adrenalectomized pups led to a further increase in pancreatic glucocorticoid-binding capacity above that due to adrenalectomy alone. When both T4 and corticosterone were given together to adrenalectomized pups their pancreatic glucocorticoid-binding capacities increased to levels above those in the adrenalectomized group, but lower than those in pups receiving T4 alone. Our results suggest that T4 modulates the development of rat pancreatic glucocorticoid receptors and, at least in part, acts via pathways independent of adrenal function.     

Enteropathy precedes type 1 diabetes in the BB rat

BACKGROUND AND AIMS: There is increasing evidence implicating intestinal immune responses to dietary proteins in the pathogenesis of type 1 autoimmune diabetes (T1D). Here we investigated the association between intestinal pathology and dietary factors in T1D by examining the mucosal architecture in the BB rat model. METHODS: BB control (BBc) and diabetes prone (BBdp) rats were fed either a diabetes retardant hydrolysed casein based diet or one of two cereal based diets that promote the development of diabetes. Intestinal architecture was assessed in the jejunum by microdissection, histology, and immunohistology, and by measuring peroxidase activity and brush border invertase levels. RESULTS: Enteropathy was present in BBdp rats soon after weaning, as assessed by increases in crypt length and in the proliferative activity of crypt epithelial cells in the jejunum, and this remained constant until 120 days of age. There was also a decrease in invertase activity, as well as increased numbers of intraepithelial lymphocytes, increased levels of mucosal peroxidase activity, and infiltration of the mucosa by CD4(+) T lymphocytes. Equivalent enteropathy was present at all times in BBdp rats and was not influenced by the nature of the diet or by thymectomy at three weeks at age, procedures which prevent the development of diabetes. CONCLUSION: Enteropathy is a consistent feature in the diabetes prone BB rat but it precedes the onset of insulitis and appears to be due to mechanisms distinct from those which cause diabetes. The beneficial effects of the diabetes retardant hydrolysed casein diet on diabetes are not due to an effect on intestinal architecture per se but mucosal damage may be necessary for the development of autoreactivity in the pancreas.     

Regulation of PKR and IRF-1 during hepatitis C virus RNA replication

The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.     

Distribution of intracellular and secreted surfactant during postnatal rat lung development

Pulmonary surfactant prevents alveolar collapse via reduction of surface tension. In contrast to human neonates, rats are born with saccular lungs. Therefore, rat lungs serve as a model for investigation of the surfactant system during postnatal alveolar formation. We hypothesized that this process is associated with characteristic structural and biochemical surfactant alterations. We aimed to discriminate changes related to alveolarization from those being either invariable or follow continuous patterns of postnatal changes. Secreted active (mainly tubular myelin (tm)) and inactive (unilamellar vesicles (ulv)) surfactant subtypes as well as intracellular surfactant (lamellar bodies (lb)) in type II pneumocytes (PNII) were quantified before (day (d) 1), during (d 7), at the end of alveolarization (d 14), and after completion of lung maturation (d 42) using electron microscopic methods supplemented by biochemical analyses (phospholipid quantification, immunoblotting for SP-A). Immunoelectron microscopy determined the localization of surfactant protein A (SP-A). (1) At d 1 secreted surfactant was increased relative to d 7-42 and then decreased significantly. (2) Air spaces of neonatal lungs comprised lower fractions of tm and increased ulv, which correlated with low SP-A concentrations in lung lavage fluid (LLF) and increased respiratory rates, respectively. (3) Alveolarization (d 7-14) was associated with decreasing PNII size although volume and sizes of Lb continuously increased. (4) The volume fractions of Lb correlated well with the pool sizes of phospholipids in lavaged lungs. Our study emphasizes differential patterns of developmental changes of the surfactant system relative to postnatal alveolarization.     

Effect of dietary vitamin E on the vitamin E status in the BB rat during development and after the onset of diabetes

Weanling diabetes-prone BB rats were fed AIN-76 diets containing high (HE, 1 g/kg diet), basal (NE, 0.2 g/kg) or low (LE, trace) vitamin E and were killed at 21, 42 or 60 days of age. Plasma and tissues (adrenals, pancreas, spleen, thymus, liver, brown and white adipose tissue, muscle and testes) were analysed for vitamin E. Vitamin E levels reflected the level in the diet and no diabetic animals were detected at these times. In a second experiment, a total of 90 diabetes-prone BB rats were kept on diets LE and HE for 6 months or until they became diabetic. 11/45 on LE and 5/45 on HE became diabetic. Again, plasma and tissue levels of vitamin E reflected the levels in the diet with the exception of the thymus of diabetic rats fed the high vitamin E diet. Thymus vitamin E levels (microgram/g tissue) were 1.8 and 1.2 in LE-fed diabetics and asymptomatic rats, respectively; and 22.7 and 49.5 in HE-fed diabetics and asymptomatic rats, respectively. The last 2 values were significantly different (p less than 0.005). There were no other differences in plasma or tissue levels of vitamin E in these groups of animals. These findings suggest that high dietary vitamin E may decrease the incidence of diabetes in animals which are able to accumulate sufficient amounts of the vitamin in the thymus. Since the thymus plays a key role in the maturation of T cell populations, which appear to be altered in this disease, it seems possible that the protective effect may be exerted at this level.     

长链非编码RNA在肺癌中的调控机制

长链非编码RNA是一类长度大于200个核苷酸,没有蛋白质编码功能的RNA。因其在肿瘤发生、发展过程中起到重要的调控作用而备受关注。肺癌是迄今发病率及病死率最高的恶性肿瘤。尽管近年来部分肺癌患者的生存期及生活质量有所改善,但其总体预后仍然不佳。因此,积极寻找更加有效的诊断及治疗方法已成为肺癌治疗工作者的严峻任务。了解长链非编码RNA在肺癌发生、发展过程中的作用及其分子机制,将为肺癌的早期诊断及治疗提供新的思路。     

Vitamin D and bone mineral homeostasis during pregnancy in the diabetic BB rat

Vitamin D and bone mineral metabolism during pregnancy were studied in 17 diabetic and 13 control BB rats. On day 21 of pregnancy, reduced mean levels of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3; 56.9 vs. 97.9 pg/ml; P less than 0.0001] and vitamin D-binding protein (304 vs. 482 micrograms/ml; P less than 0.0001) were found in the diabetic rats, while the free 1,25-(OH)2D3 concentration was not different from the control level. Total plasma calcium and total plasma protein concentrations were also significantly decreased in the diabetic group, but the calculated diffusible calcium was not significantly lower. Calcium and phosphorus urinary excretion were increased in the diabetic rats. There was no difference in bone mineral content. The fetuses of the diabetic BB rat had a lower body weight and were hypoinsulinemic. Both 1,25-(OH)2D3 (41.3 vs. 54.7 pg/ml; P less than 0.01) and vitamin D-binding protein (80 vs. 123 micrograms/ml; P less than 0.001) were decreased in the fetuses of diabetic rats, but the free 1,25-(OH)2D3 concentration was slightly but significantly (6.96 vs. 5.54; P less than 0.05) increased. We observed that the fetuses of diabetic rats had fewer ossification centers, counted with the Alizarin Red S staining method. The fetal ash weight was lower in the diabetic group (16.7 vs. 26.9 mg; P less than 0.0001). In addition, the relative calcium and phosphorus, but not magnesium, content of ash was lower in the fetuses of diabetic rats. This reduced mineral content in fetuses of diabetic mothers could be implicated in the pathogenesis of early neonatal hypocalcemia in infants of diabetic mothers.     

Regulation of the procoagulant activity within the bronchoalveolar compartment of normal human lung

The nature of the procoagulant activity of normal bronchoalveolar fluid was examined both qualitatively and quantitatively. Unconcentrated, cell-free lavage freshly obtained from normal volunteers clotted normal plasma in a mean of 84 +/- 20 s. The procoagulant activity was initiated by Factor VII-tissue factor complexes as judged by differential activity in various plasmas genetically deficient in single clotting factors, by neutralization of the procoagulant activity with antibodies to either Factor VII or tissue factor, and by a Factor X activation assay. Preincubation of the lavage with calcium was required to demonstrate Factor VII activity in unconcentrated samples. The cell-free fluid contained about 8,500 thromboplastin units/mg protein, equivalent to a third of the thromboplastin standard and indicating high amounts of cofactor. Quantitation of Factor VII was estimated by functional analysis in coagulation and amidolytic assays with reference to dilutions of normal plasma of known Factor VII concentration. When lavage and diluted plasma were adjusted to yield equivalent amidolytic activities, the average ratio of the Factor VII-clotting activity of the alveolar fluid relative to plasma Factor VII was 19 +/- 7, suggesting the presence of Factor VIIa in lavage. In contrast to previous reports with serum or activated plasma, immunoblots of concentrated lavage revealed only single-chain Factor VII, and 125I-Factor VII added to the fluid was not converted to 125I-Factor VIIa, suggesting a unique control mechanism in the lung compartment which differs from plasma. When equivalent Factor VII amidolytic activities in diluted plasma and cell-free lavage were compared, the rates of Factor Xa formation were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin content of normal and of bleomycin-fibrotic rat lung

A large proportion of parenchymal lung is composed of cells with an abundance of actin-containing cytoplasmic microfilaments. This may explain the substantial contractile capability of the tissue, which it appears cannot be ascribed totally to vascular or airway smooth muscle. Parenchymal contractility is increased in bleomycin-induced fibrosis, together with an increase in microfilaments and actin- and myosin-directed immunofluorescence. The present biochemical studies indicate that at least 10% of detergent-extractable protein from peripheral lung is actin. Approximately 71% of this actin is polymerized and 29% is monomeric, on the basis of differential ultracentrifugation. Isoelectric focusing and tryptic peptide analysis show that nonmuscle actin types predominate in the parenchyma, of which approximately 78% is beta-nonmuscle actin and 19% is gamma-nonmuscle actin, together with approximately 3% gamma-smooth muscle actin. Total actin as a percentage of extractable protein was not increased significantly in lungs of rats 4 or 8 wk after bleomycin instillation. Thus, actin per total lung is increased only in proportion to increased total lung weight. There is, in addition, no detectable shift in the beta/gamma actin ratio in the fibrotic lung or increase in percentage of smooth muscle actin. There is, however, a significant 16% decrease in monomeric G-actin and a commensurate significant increase in the percentage of polymerized or F-actin. Therefore, increased contractility and actin-specific immunofluorescence characteristic of fibrotic lung does not appear to be due to increases in total actin but rather to increases in its degree of polymerization, as is found in a variety of remodeling tissues. The consequence of these changes in contractile protein organization to lung function requires further investigation.     

Postnatal development of rat lung following retarded fetal lung growth

Postnatal lung development was examined in rats born with smaller than normal lungs after either prenatal exposure to glucocorticoid or to an inhibitor of collagen synthesis. At birth, treated animals had lower than normal lung weights, lung to body weight ratios, hydroxyproline (HYP) levels, total DNA; and rates of DNA synthesis. Rats exposed to steroid showed a rapid recovery in growth during the normal postnatal cell proliferative phase from 4 to 11 days, though collagen levels did not return to normal until 3 weeks of age. Rats exposed to a prenatal proline analog showed a much slower rise in lung weight and total DNA, and these levels were still much below normal at 2-3 weeks when the cell proliferation phase was completed. Levels of disaturated phosphatidylcholine were significantly below normal up to 11 days, whereas total HYP was significantly reduced and less fibrillar collagen was seen in the lung throughout the study. The results indicate that the smaller but mature lungs at birth after antenatal steroid show a growth rebound and quickly become structurally normal. In contrast, inhibition of fibroblast growth and collagen deposition produces small lungs at birth, which continue to show inhibited growth and development at least up to 3 weeks of age, when the cell division phase is over.