Pregnancy-associated plasma protein-A regulates myoblast proliferation and differentiation through an insulin-like growth factor-dependent mechanism

Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAPP-A increased their proliferation in a dose- and time-dependent manner. Addition of exogenous PAPP-A also increased the myotube formation and the activity of creatine kinase in C2C12 cultures. Transient overexpression of the full-length PAPP-A-(1-1547), but not truncated protease-inactive N-terminal PAPP-A-(1-920) or C-terminal PAPP-A-(1100-1547), significantly enhanced the proliferation of C2C12 myoblasts. In vitro and in situ experiments demonstrated that PAPP-A cleaves insulin-like growth factor-binding protein (IGFBP)-2, but not IGFBP-3, in the conditioned medium of C2C12 myoblasts. Overexpression of PAPP-A led to degradation of the IGFBP-2 produced by C2C12 myoblasts and increased free IGF-I concentrations without affecting total IGF-I concentrations. Addition of protease-resistant IGFBP-4 completely abolished the PAPP-A-induced proliferation of C2C12 myoblasts. Our results demonstrate that 1) PAPP-A increases the proliferation and differentiation of myoblasts, 2) the stimulatory effect of PAPP-A on myogenesis is governed by its proteolytic activity, and 3) PAPP-A promotes skeletal myogenesis by increasing the amount of free IGFs via specific degradation of IGFBP-2 produced by myoblasts.     

Developmental changes in amniotic and allantoic fluid insulin-like growth factor (IGF)-I and -II concentrations of avian embryos

In the literature, IGFs in the developing embryo are usually determined by blood serum concentrations. For this study, IGF-I/-II was quantified in the amniotic and allantoic fluids of fertile commercial broiler chicken (Gallus domesticus) (n=222), Pekin duck (Anas platyrhyncha) (n=250), and turkey (Meleagridis gallopavo) eggs (n= 200) during incubation. Amniotic and allantoic fluids were collected from embryos starting at 6 days of incubation for chickens and 8 days of incubation for ducks and turkeys. IGF concentrations within the fluids were determined by radioimmunoassay. Chicken amniotic IGF-I concentration at stage 29 of development was significantly higher (P< or =0.05) than the duck or turkey. At stage 36 of development the concentration of IGF-II in the amniotic fluid was 2.8 times greater in the chicken versus the duck (P< or =0.05) and 2 times greater than in the turkey (P< or =0.05). Within species, chicken IGF-I concentration in the amniotic fluid had a cubic trend (P< or =0.001), duck IGF-I increased linearly (P< or =0.001), and turkey concentrations declined quadratically (P< or =0.001) throughout development. In all species, the IGF-II concentration was higher than the IGF-I concentration in the amniotic and allantoic fluids.     

IGF-I and NEFA concentrations in fetal fluids of term pregnancy dogs

Insulin-like growth factor-I (IGF-I) and non-esterified fatty acids (NEFA) play an essential role in fetal growth and development. To date, fetal fluids IGF-I and NEFA levels at term canine pregnancy are unknown and could be related to the neonatal development and breed size. For these reasons, the aims of the present study were as follows: (1) to evaluate IGF-I and NEFA concentrations in fetal fluids collected from normally developed and viable newborn puppies born at term of normal pregnancies; (2) to assess possible differences between IGF-I and NEFA levels in amniotic compared with allantoic fluid; (3) to detect possible relationship between breed body size and IGF-I and NEFA amniotic and allantoic concentrations; (4) to evaluate possible differences in IGF-I fetal fluids levels between male and female puppies; and (5) to assess possible correlations between the two hormones in each type of fluid. The study enrolled 25 pure breed bitches submitted to elective Cesarean section at term because of the high risk of dystocia or previous troubles at parturition. At surgery, amniotic and allantoic fluids were collected and assayed for IGF-I and NEFA. IGF-I and NEFA amounts in both amniotic and allantoic fluids of different breed size bitches (small: ≤10 kg; medium: 11–25 kg; large: 26–40 kg) were detected, as well as the effect of gender on IGF-I levels. On a total of 73 amniotic and 76 allantoic samples collected by normal, viable, and mature newborns, the mean IGF-I concentration was significantly higher in amniotic than in allantoic fluid in all three groups, but the amniotic IGF-I levels were significantly lower in small and medium size bitches when compared with large ones. No significant differences were found in allantoic IGF-I concentrations among size groups. A significant effect of the puppy gender on IGF-I content in both fetal fluids was not reported. Regarding NEFA, in all the three groups, the mean NEFA concentration did not significantly differ between amnion and allantois, but in both fetal fluids, higher NEFA levels were detected in samples belonging to small breeds when compared with medium and large. These data strongly indicated that, also in the dog, a relation between fetal fluids IGF-I and NEFA concentrations and breed size exists. Further research is needed to elucidate the possible role of IGF-I and NEFA in the pathologic conditions related to canine fetal growth.     

Differential responses of IGF-I molecular complexes to military operational field training.

Insulin-like growth factor (IGF) I and IGF binding proteins (IGFBPs) modulate metabolic activity and tissue repair and are influenced by nutritional status. IGF-I circulates in free, ternary [IGF-I + IGFBP-3 + acid labile subunit (ALS)], and binary (IGF-I + IGFBP) molecular complexes, and the relative proportions regulate IGF-I extravascular shifting and bioavailability. This study examined the hypothesis that sustained physical activity and sleep deprivation superimposed on a short-term energy deficit would alter the IGFBP concentrations and alter the proportions of IGF-I circulating in ternary vs. binary molecular complexes. Components of the IGF-I system (total and free IGF-I; IGFBP-1, -3, and ALS; nonternary IGF-I and IGFBP-3), biomarkers of metabolic and nutritional status (transferrin, ferritin, prealbumin, glucose, free fatty acids, glycerol, beta-hydroxybutyrate), and body composition were measured in 12 men (22 +/- 3 yr, 87 +/- 8 kg, 183 +/- 7 cm, 20 +/- 5% body fat) on days 1, 3, and 4 during a control and experimental (Exp) period. During Exp, subjects performed prolonged work (energy expenditure of approximately 4500 kcal/day) with caloric (1600 kcal/day) and sleep (6.2 h total) restriction. IGF-I and IGFBP-3 were measured by immunoassay before and after immunoaffinity depletion of ALS-based complexes (i.e., ternary complex removal). Exp produced losses in body mass (-3.0%), lowered total IGF-I (-24%), free IGF-I (-42%), IGFBP-3 (-6%), nonternary IGF-I (-27%), and IGFBP-3 (-16%), and increased IGFBP-1 (256%). No Exp effects were observed for ALS. No changes were observed in the proportion of IGF-I circulating in free ( approximately 1.2%), ternary ( approximately 87.4%), or nonternary ( approximately 11.4%) molecular complexes. During Exp, glucose concentrations were lower on day 3, but days 1 and 4 were statistically similar. In conclusion, during a short-term energy deficit in young, healthy men, 1). IGF-I system components differentially respond (both in direction and magnitude) to a given metabolic perturbation and 2). the relative proportion of IGF-I sequestered in ternary vs. nonternary molecular complexes appears to be well maintained.     

Genetic polymorphisms of antioxidant enzymes as risk factors for oxidative stress-associated complications in preterm infants

We aimed to identify specific polymorphisms of genes encoding for superoxide dismutase (SOD) 1 (cytoplasmic Cu/ZnSOD), SOD2 (mitochondrial MnSOD), SOD3 (extracellular Cu/ZnSOD) and CAT in a cohort of preterm infants and correlate their presence to the development of respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular haemorrhage (IVH) and retinopathy of prematurity (ROP). We carried out a retrospective study to evaluate the allele frequency and the genotype distribution of polymorphisms of SODs and CAT in a population of preterm neonates (n = 152) with a gestational age ≤ 28 weeks according to the presence or absence of RDS, BPD, IVH and ROP. Moreover, we evaluated through the haplotype reconstruction analysis whether combinations of the selected polymorphisms are related to the occurrence of RDS, BPD, IVH and ROP. We found that rs8192287 SOD3 polymorphism is an independent protective factor for all grade IVH, while rs4880 and rs5746136 SOD2 polymorphisms are associated with a lower gestational age (rs4880, rs5746136) and birth weight (rs4880). Haplotypes reconstruction showed that SOD1 (GG) decreased the risk of RDS, IVH and ROP; SOD2 (GT) increased the risk of BPD and decreased the risk of RDS, IVH and ROP; SOD3 (TGC) decreased the risk of BPD and IVH; and 4) CAT (CTC) decreased the risk of RDS. The rs8192287 SOD3 polymorphism is per se an independent predictor of a decreased risk of developing IVH. Different SOD2 polymorphisms are associated per se with a lower gestational age and/or birth weight, and haplotypes of SOD1, SOD3 and CAT genes may be independent protecting or risk markers for prematurity complications.     

Correlation of urinary inflammatory and oxidative stress markers in very low birth weight infants with subsequent development of bronchopulmonary dysplasia

Currently, bronchopulmonary dysplasia (BPD) occurs almost exclusively in pre-term infants. In addition to prematurity, other factors like oxygen toxicity and inflammation can contribute to the pathogenesis. This study aimed to compare urinary inflammatory and oxidative stress markers between the no/mild BPD group and moderate/severe BPD group and between BPD cases with significant early lung disease like respiratory distress syndrome (RDS) ('classic' BPD) and with minimal early lung disease ('atypical' BPD). A total of 60 patients who were a gestational age < 30 weeks or a birth weight < 1250 g were included. Urine samples were obtained on the 1(st), 3(rd) and 7(th) day of life and measured the levels of leukotriene E(4) (LTE(4)) and 8-hydroxydeoxyguanosine (8-OHdG). The 8-OHdG values on the 3(rd) day showed significant correlation to duration of mechanical ventilation. The 8-OHdG levels on the 7(th) day were the independent risk factor for developing moderate/severe BPD. In 'classic' BPD, the 8-OHdG values on the 3(rd) day were higher than those of 'atypical' BPD. In 'atypical' BPD, the LTE(4) values on the 7(th) day were higher than the values in 'classic' BPD. These results suggest that oxidative DNA damage could be the crucial mechanism in the pathogenesis of current BPD and the ongoing inflammatory process could be an important mechanism in 'atypical' BPD.     

Genetic polymorphisms of antioxidant enzymes as risk factors for oxidative stress-associated complications in preterm infants

Abstract

We aimed to identify specific polymorphisms of genes encoding for superoxide dismutase (SOD) 1 (cytoplasmic Cu/ZnSOD), SOD2 (mitochondrial MnSOD), SOD3 (extracellular Cu/ZnSOD) and CAT in a cohort of preterm infants and correlate their presence to the development of respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular haemorrhage (IVH) and retinopathy of prematurity (ROP). We carried out a retrospective study to evaluate the allele frequency and the genotype distribution of polymorphisms of SODs and CAT in a population of preterm neonates (n =?152) with a gestational age ≤?28 weeks according to the presence or absence of RDS, BPD, IVH and ROP. Moreover, we evaluated through the haplotype reconstruction analysis whether combinations of the selected polymorphisms are related to the occurrence of RDS, BPD, IVH and ROP. We found that rs8192287 SOD3 polymorphism is an independent protective factor for all grade IVH, while rs4880 and rs5746136 SOD2 polymorphisms are associated with a lower gestational age (rs4880, rs5746136) and birth weight (rs4880). Haplotypes reconstruction showed that SOD1 (GG) decreased the risk of RDS, IVH and ROP; SOD2 (GT) increased the risk of BPD and decreased the risk of RDS, IVH and ROP; SOD3 (TGC) decreased the risk of BPD and IVH; and 4) CAT (CTC) decreased the risk of RDS. The rs8192287 SOD3 polymorphism is per se an independent predictor of a decreased risk of developing IVH. Different SOD2 polymorphisms are associated per se with a lower gestational age and/or birth weight, and haplotypes of SOD1, SOD3 and CAT genes may be independent protecting or risk markers for prematurity complications.     

Nutrition of preterm infants in relation to bronchopulmonary dysplasia

Background

The pathogenesis of bronchopulmonary dysplasia (BPD) is multifactorial. In addition to prenatal inflammation, postnatal malnutrition also affects lung development.

Methods

A retrospective study was performed to analyse during the first two weeks of life the total, enteral and parenteral nutrition of premature infants (<31 weeks, birth weight ≤1500 g) born between 08/04 and 12/06.

Results

Ninety-five premature infants were analysed: 26 with BPD (27 ± 1 weeks) and 69 without BPD (28 ± 1 weeks). There was no statistical significant difference in the total intake of fluids, calories, glucose or protein and weight gain per day in both groups. The risk of developing BPD was slightly increased in infants with cumulative caloric intake below the minimal requirement of 1230 kcal/kg and a cumulative protein intake below 43.5 g/kg. Furthermore, the risk of developing BPD was significantly higher when infants had a cumulative fluid intake above the recommended 1840 ml/kg. In infants who developed BPD, the enteral nutrition was significantly lower than in non-BPD infants [456 ml/kg (IQR 744, 235) vs. 685 (IQR 987, 511)]. Infants who did not develop BPD reached 50% of total enteral feeding significantly faster [9.6 days vs. 11.5].

Conclusions

Preterm infants developing BPD received less enteral feeding, even though it was well compensated by the parenteral nutrient supply. Data suggest that a critical minimal amount of enteral feeding is required to prevent development of BPD; however, a large prospective clinical study is needed to prove this assumption.

     

Stimulation of HIF-1alpha, HIF-2alpha, and VEGF by prolyl 4-hydroxylase inhibition in human lung endothelial and epithelial cells

Diminished alveolar and vascular development is characteristic of bronchopulmonary dysplasia (BPD) affecting many preterm newborns. Hypoxia promotes angiogenic responses in developing lung via, for example, vascular endothelial growth factor (VEGF). To determine if prolyl 4-hydroxylase (PHD) inhibition could augment hypoxia-inducible factors (HIFs) and expression of angiogenic proteins essential for lung development, HIF-1alpha and -2alpha proteins were assessed in human developing and adult lung microvascular endothelial cells and alveolar epithelial-like cells treated with either the HIF-PHD-selective inhibitor PHI-1 or the nonselective PHD inhibitors dimethyloxaloylglycine (DMOG) and deferoxamine (DFO). PHI-1 stimulated HIF-1alpha and -2alpha equally or more effectively than did DMOG or DFO, enhanced VEGF release, and elevated glucose consumption, whereas it was considerably less cytotoxic than DMOG or DFO. Moreover, VEGF receptor Flt-1 levels increased, whereas KDR/Flk-1 decreased. PHI-1 treatment also increased PHD-2, but not PHD-1 or -3, protein. These results provide proof of principle that HIF stimulation and modulation of HIF-regulated angiogenic proteins through PHI-1 treatment are feasible, effective, and nontoxic in human lung cells, suggesting the use of PHI-1 to enhance angiogenesis and lung growth in evolving BPD.     

Serum levels of total and free IGF-I and IGFBP-3 are increased and maintained in long-term training.

The goals of this study were to determine whether the long-term training regimens experienced by competitive collegiate swimmers would result in altered levels of total and free serum insulin-like growth factor I (IGF-I) as well as IGF-binding proteins (BP) IGFBP-1 and -3. Two male (Teams 1M and 2M) and one female (Team 2F) teams were studied at the start of training, after 2 mo of training, after 4 mo (2-4 mo had the highest volume of training), after 5 mo (near the end of tapering; only for Team 1M), and several days after training was over. For Team 1M, total IGF-I concentrations were increased by 76% after 4 mo and were subsequently maintained at this level. Total IGF-I responses were more variable for Teams 2F and 2M. Free IGF-I levels were increased nearly twofold for all teams at 2 mo and were maintained or increased further with subsequent training. Only the levels of free IGF-I for Team 1M returned to pretraining values after training had ended. Training had little effect on IGFBP-1 levels. For all teams, serum IGFBP-3 was elevated by 4 mo of training (for Team 2F it was increased at 2 mo) by 30-97% and remained at these higher levels thereafter. The ratio of total IGF-I to IGFBP-3 was not increased by training in any group. These data indicate that serum levels of total and free IGF-I and total IGFBP-3 can be increased with intense training and maintained with reduced training (tapering). The findings show that changes in free IGF-I levels are not accounted for by alterations in the total IGF-I/IGFBP-3 complex or in IGFBP-3 levels and indicate that there are other important determinants of free IGF-I.     

Mesenchymal stem cells from different sources show distinct therapeutic effects in hyperoxia-induced bronchopulmonary dysplasia in rats

Mesenchymal stem cells (MSCs) have been shown as an effective medicinal means to treat bronchopulmonary dysplasia (BPD). The widely used MSCs were from Wharton's jelly of umbilical cord (UC-MSCs) and bone marrow (BM-MSCs). Amniotic fluid MSCs (AF-MSCs) may be produced before an individual is born to treat foetal diseases by autoplastic transplantation. We evaluated intratracheal (IT) MSCs as an approach to treat an hyperoxia-induced BPD animal model and compared the therapeutic effects between AF-, UC- and BM-MSCs. A BPD animal model was generated by exposing newborn rats to 95% O₂. The continued stress lasted 21 days, and the treatment of IT MSCs was conducted for 4 days. The therapeutic effects were analysed, including lung histology, level of inflammatory cytokines, cell death ratio and state of angiogenesis, by sacrificing the experimental animal at day 21. The lasting hyperoxia stress induced BPD similar to the biological phenotype. The treatment of IT MSCs was safe without deaths and normal organ histopathology. Specifically, the treatment was effective by inhibiting the alveolar dilatation, reducing inflammatory cytokines, inducing angiogenesis and lowering the cell death ratio. AF-MSCs had better therapeutic effects compared with UC-MSCs in relieving the pulmonary alveoli histological changes and promoting neovascularization, and UC-MSCs had the best immunosuppressive effect in plasma and lung lysis compared with AF-MSCs and BM-MSCs. This study demonstrated the therapeutic effects of AF-, UC- and BM-MSCs in BPD model. Superior treatment effect was provided by antenatal MSCs compared to BM-MSC in a statistical comparison.     

Phosphodiesterase 4 inhibition attenuates persistent heart and lung injury by neonatal hyperoxia in rats

Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.     

Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles: a control mechanism for selection of dominant follicles

This review summarizes evidence for the role of proteolytic enzymes that degrade and inactivate insulin-like growth factor binding proteins (IGFBP) during follicular development in mammals. In some species (e.g., bovine), evidence indicates that decreases in IGFBP-4 and -5 levels in estrogen-dominant preovulatory follicles are likely due, in part, to increased protease activity, whereas lower levels of IGFBP-2 are not due to increased proteolysis. Increased IGFBP-4 and -5 protease along with lower amounts of IGFBP-4 binding activity and greater amounts of free IGF-I are some of the earliest developmental changes documented in bovine growing antral follicles. This protease activity has recently been ascribed to serine metalloprotease(s), including pregnancy-associated plasma protein-A (PAPP-A), which was first detected in human follicular fluid nearly 20 yr ago. Other recent studies verified the presence of PAPP-A mRNA in granulosa cells of humans, monkeys, cattle, mice, and pigs. Increases in the amount of PAPP-A mRNA in granulosa cells during follicular development occurs in some but not all species, indicating that other proteases or protease inhibitors may be involved in IGFBP degradation. Whether the hormonal control of PAPP-A production/activity by the ovary differs between monotocous and polytocous animals will require further study. These protease-induced decreases in IGFBP-4 and -5 likely cause increased levels of bioavailable (or free) IGFs that stimulate steroidogenesis and mitogenesis in developing dominant follicles, which ultimately prepare the follicle(s) and oocyte(s) for successful ovulation and fertilization.     

Correlation of urinary inflammatory and oxidative stress markers in very low birth weight infants with subsequent development of bronchopulmonary dysplasia

Abstract

Currently, bronchopulmonary dysplasia (BPD) occurs almost exclusively in pre-term infants. In addition to prematurity, other factors like oxygen toxicity and inflammation can contribute to the pathogenesis. This study aimed to compare urinary inflammatory and oxidative stress markers between the no/mild BPD group and moderate/severe BPD group and between BPD cases with significant early lung disease like respiratory distress syndrome (RDS) (‘classic’ BPD) and with minimal early lung disease (‘atypical’ BPD). A total of 60 patients who were a gestational age < 30 weeks or a birth weight < 1250 g were included. Urine samples were obtained on the 1^st, 3^rd and 7^th day of life and measured the levels of leukotriene E₄ (LTE₄) and 8-hydroxydeoxyguanosine (8-OHdG). The 8-OHdG values on the 3^rd day showed significant correlation to duration of mechanical ventilation. The 8-OHdG levels on the 7^th day were the independent risk factor for developing moderate/severe BPD. In ‘classic’ BPD, the 8-OHdG values on the 3^rd day were higher than those of ‘atypical’ BPD. In ‘atypical’ BPD, the LTE₄ values on the 7^th day were higher than the values in ‘classic’ BPD. These results suggest that oxidative DNA damage could be the crucial mechanism in the pathogenesis of current BPD and the ongoing inflammatory process could be an important mechanism in ‘atypical’ BPD.     

Spontaneous resolution of pulmonary edema caused by short periods of cyclic overinflation.

Mechanical ventilation with high or even moderate peak inspiratory pressure produces pulmonary permeability edema. Besides the level of overinflation, duration may affect both severity and type of edema. We studied the effect of 2 min of 35-mmHg peak pressure mechanical ventilation (HV) on microvascular permeability and deep lung fluid balance in rats. It resulted in increased extravascular lung water (+50%), bloodless dry lung weight (+25%), and albumin uptake in lungs (+450%). The increase in dry lung weight and albumin uptake compared with that of lung water suggested major permeability alterations. Ultrastructural examination showed the presence of numerous endothelial blebs. Epithelial lining fluid (ELF) volume, its potassium and protein concentrations, and cellular composition were assessed by bronchoalveolar lavage. There was an increase in ELF volume (+180%), a decrease in ELF potassium concentration (-50%), and an increase in ELF protein content (+76%). A few blood cells were recovered, suggesting the presence of a few large epithelial breaks. Some animals were allowed to recover for periods less than or equal to 180 min after HV. Extravascular lung water, dry lung weight, and albumin distribution space returned to control levels within 45 min. ELF volume diminished but remained larger than in controls, and ELF protein concentration increased probably because of alveolar fluid resorption. No further hemorrhage was observed. These results indicate that periods of HV as short as 2 min transiently alter microvascular permeability in rats.     

Respiratory tract gene transfer. Transplantation of genetically modified T-lymphocytes directly to the respiratory epithelial surface.

To evaluate the strategy for potentially treating respiratory disorders with genetically modified T-lymphocytes, the interleukin-2 (IL-2)-dependent murine T-cell line, CTLL2, was genetically altered with the Escherichia coli beta-galactosidase (beta-gal) gene (lacZ) in vitro with a retroviral vector and the modified T-cells were transplanted directly to the respiratory epithelial surface of syngeneic C57Bl/6 mice. Southern and Northern analyses confirmed that the neomycin-selected modified T-cells contained and expressed the lacZ gene. The fate of the modified T-cells (CTLL2/lacZ) was followed by flow cytometry with T-cell surface marker Thy1.2 and fluorescent beta-gal analysis. One day after transplantation (7.5 x 10(5) CTLL2/lacZ T-cells/g of body weight), 95 +/- 3% of the Thy1.2+ T-cells recovered from respiratory epithelial lining fluid (ELF) were beta-gal+. Importantly, the modified T-cells remained in the lung for some time; at 3 days, Thy1.2+ beta-gal+ T-cells represented 63 +/- 12% of ELF Thy1.2+ T-cells and 59 +/- 6% of Thy1.2+ T-cells recovered from the whole lung. At 7 days, 33 +/- 8% of the Thy 1.2+ cells in ELF and 75 +/- 6% of the Thy1.2+ cells in whole lung were Thy1.2+ beta-gal+. In contrast, the proportion of the Thy1.2+ beta-gal+ T-cells in the spleen, the major extrapulmonary lymphatic organ, never rose above 3 +/- 1% of the total Thy1.2+ cells. The number of Thy1.2+ beta-gal+ T-cells in the lung could be modified by the systemic administration of IL-2, with whole lung Thy1.2+ beta-gal+ T-cells increasing 4.6-fold 3 days after transplantation, compared with non-IL-2-treated animals. These studies suggest that direct transplantation of genetically modified T-cells into the lung is feasible and represents a viable strategy for lung-specific gene transfer.     

Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.     

Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa

In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model. The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge. Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge. Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge. Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection. Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid. Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected. When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge. No inhibition was observed by anti-CINC-1 antibody. These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P. aeruginosa.