A comparison of four different models of acute respiratory distress syndrome in sheep.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can have various causes. The study objective was to investigate whether different pathophysiologic models of ARDS would show different respiratory, cardiovascular and inflammatory outcomes. METHODS: We performed a prospective, randomized study in 27 ventilated ewes inducing ARDS using three different techniques to mimic the pulmonary causes of ARDS (ARDSp): warm saline lavage (n = 6), intratracheal hydrochloric acid (HCl; n = 6), intratracheal albumin (n = 10), and one technique to mimic an extrapulmonary cause of ARDS (ARDSexp): intravenous lipopolysaccharide (LPS iv; n = 5). ARDS was defined when PaO2 was < 15 kPa (112 mmHg) when ventilated with PEEP 10 cm H2O and FiO2 = 1.0. The effects on gas exchange were investigated by calculating the oxygenation index (OI) and the ventilation efficacy index (VEI) every 30 min for a period of 4 h. Post mortem lung lavage was performed to obtain broncho-alveolar lavage fluid (BALF) to assess lung injury and inflammation. Lung injury and inflammation were assessed by measuring the total number and differentiation of leukocytes, the concentration of protein and disaturated phospholipids, and interleukine-6 and -8 in the BALF. Histology of the lung was evaluated by measuring the mean alveolar size, alveolar wall thickness and the lung injury score system by Matute-Bello et al., as markers of lung injury. The concentration of interleukin-6 was determined in plasma, as a marker of systematic inflammation. RESULTS: The OI and VEI were most affected in the LPS iv group and thereafter the HCl group, after meeting the ARDS criteria. Diastolic blood pressure was lowest in the LPS iv group. There were no significant differences found in the total number and differentiation of leukocytes, the concentration of protein and disaturated phospholipids, or interleukin-8 in the BALF, histology of the lung and the lung injury score. IL-6 in BALF and plasma was highest in the LPS iv group, but no significant differences were found between the other groups. It took a significantly longer period of time to meet the ARDS criteria in the LPS iv group. CONCLUSIONS: The LPS model caused the most severe pulmonary and cardiovascular insufficiency. Surprisingly, there were limited significant differences in lung injury and inflammatory markers, despite the different pathophysiological models, when the clinical definition of ARDS was applied.

Global hypoxia-ischemia induced inflammation and structural changes in the preterm ovine gut which were not ameliorated by mesenchymal stem cell treatment.

Perinatal asphyxia, a condition of impaired gas exchange during birth, leads to fetal hypoxia-ischemia (HI) and is associated with postnatal adverse outcomes including intestinal dysmotility and necrotizing enterocolitis (NEC). Evidence from adult animal models of transient, locally-induced intestinal HI has shown that inflammation is essential in HI-induced injury of the gut. Importantly, mesenchymal stem cell (MSC) treatment prevented this HI-induced intestinal damage. We therefore assessed whether fetal global HI induced inflammation, injury and developmental changes in the gut and whether intravenous MSC administration ameliorated these HI-induced adverse intestinal effects. In a preclinical ovine model, fetuses were subjected to umbilical cord occlusion (UCO), with or without MSC treatment, and sacrificed 7 days after UCO. Global HI increased the number of myeloperoxidase positive cells in the mucosa, upregulated mRNA levels of interleukin (IL)-1ß and IL-17 in gut tissue and caused T-cell invasion in the intestinal muscle layer. Intestinal inflammation following global HI was associated with increased Ki67+ cells in the muscularis and subsequent muscle hyperplasia. Global HI caused distortion of glial fibrillary acidic protein immunoreactivity in the enteric glial cells and increased synaptophysin and serotonin expression in the myenteric ganglia. Intravenous MSC treatment did not ameliorate these HI-induced adverse intestinal events. Global HI resulted in intestinal inflammation and enteric nervous system abnormalities which are clinically associated with postnatal complications including feeding intolerance, altered gastrointestinal transit and NEC. The intestinal histopathological changes were not prevented by intravenous MSC treatment directly after HI, indicating that alternative treatment regimens for cell-based therapies should be explored.

The effects of dexamethasone and oxygen in ventilated adult sheep with early phase acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threating condition with high morbidity and mortality. Inflammation is the main factor in the pathogenesis of ARDS. Therefore systemic corticosteroids are a rational therapeutic approach, but the effect of corticosteroids is still unclear. In this study, we looked at the effects of corticosteroids in ventilated sheep with ARDS, induced by lung lavage. METHODS: We performed a prospective, randomised study in 64 ventilated sheep with ARDS, to evaluate the effect of corticosteroids and oxygen concentration on gas exchange and lung injury. Oxygenation index (OI) and ventilation efficacy index (VEI) were calculated to evaluate gas exchange. Lung injury was assessed by inflammatory response in broncho-alveolar lavage fluid (BALF) and plasma and histology of the lung. RESULTS: OI, VEI, lung inflammation, surfactant production, or lung histology was not influenced by corticosteroids. In the 100 % oxygen groups, OI was higher and total number of cells and disaturated phospholipids were lower in BALF. CONCLUSION: Our study showed that corticosteroids did not influence inflammation in early phase ARDS and that hyperoxia aggravated lung injury which could not be modulated by dexamethasone in early phase ARDS.

Alveolocapillary model system to study alveolar re-epithelialization.

In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process.

Comparison of recruitment manoeuvres in ventilated sheep with acute respiratory distress syndrome.

BACKGROUND: Recruitment manoeuvres are widely used in clinical practice to open the lung and prevent lung injury by derecruitment, although the evidence is still discussed. In this study two different recruitment manoeuvres were compared to no recruitment manoeuvres (control) in ventilated sheep with acute respiratory distress syndrome (ARDS), induced by lung lavage. METHODS: We performed a prospective, randomised study in 26 ventilated sheep with ARDS, to evaluate the effect of two different recruitment manoeuvres on gas exchange, blood pressure and lung injury. The two different recruitment manoeuvres, the high pressure recruitment manoeuvre (HPRM), with high peak pressure, and the smooth and moderate recruitment manoeuvre (SMRM), with lower peak pressure, were compared to controls (no recruitment) after disconnection. Oxygenation index and ventilation efficacy index were calculated to evaluate gas exchange. Lung injury was assessed by inflammatory response in broncho-alveolar lavage fluid (BALF) and blood and histology of the lung. RESULTS: Oxygenation index improved significantly after both recruitment manoeuvres compared with controls, but no significant difference was found between the recruitment manoeuvres. Blood pressure decreased after HPRM but not after SMRM. HPRM induced a higher number of total cells and more neutrophils in the BALF. In the histology of the lung, mean alveolar size was increased in the dorsocranial region of the lung of SMRM compared to controls. CONCLUSION: Recruitment manoeuvres improved oxygenation, but SMRM was superior, with respect to hemodynamics and pulmonary inflammation, in ventilated sheep suffering from ARDS induced by lung lavage.

Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey.

Many whey proteins, peptides and protein-derived amino acids have been suggested to improve gut health through their anti-oxidant, anti-microbial, barrier-protective and immune-modulating effects. Interestingly, although the degree of hydrolysis influences peptide composition and, thereby, biological function, this important aspect is often overlooked. In the current study, we aimed to investigate the effects of whey protein fractions with different degrees of enzymatic hydrolysis on the intestinal epithelium in health and disease with a novel 2D human intestinal organoid (HIO) monolayer model. In addition, we aimed to assess the anti-microbial activity and immune effects of the whey protein fractions. Human intestinal organoids were cultured from adult small intestines, and a model enabling apical administration of nutritional components during hypoxia-induced intestinal inflammation and normoxia (control) in crypt-like and villus-like HIO was established. Subsequently, the potential beneficial effects of whey protein isolate (WPI) and two whey protein hydrolysates with a 27.7% degree of hydrolysis (DH28) and a 50.9% degree of hydrolysis (DH51) were assessed. In addition, possible immune modulatory effects on human peripheral immune cells and anti-microbial activity on four microbial strains of the whey protein fractions were investigated. Exposure to DH28 prevented paracellular barrier loss of crypt-like HIO following hypoxia-induced intestinal inflammation with a concomitant decrease in hypoxia inducible factor 1 alpha (HIF1α) mRNA expression. WPI increased Treg numbers and Treg expression of cluster of differentiation 25 (CD25) and CD69 and reduced CD4+ T cell proliferation, whereas no anti-microbial effects were observed. The observed biological effects were differentially mediated by diverse whey protein fractions, indicating that (degree of) hydrolysis influences their biological effects. Moreover, these new insights may provide opportunities to improve immune tolerance and promote intestinal health.

Surfactant protein A influences reepithelialization in an alveolocapillary model system.

PURPOSE: Restoring the barrier integrity of the alveolar epithelium after injury is pivotal. In the current study, we evaluated the effects of surfactant, surfactant protein A (SP-A), transforming growth factor ß (TGF-ß), and analogues of SP-A on alveolar epithelial repair. Additionally, we assessed the influence of microvascular endothelial cells on reepithelialization. METHODS: Repair was studied in an in vitro model system consisting of a bilayer coculture of A549 and human pulmonary microvascular endothelial cells (HPMECs), which stably expressing fluorescent proteins. The epithelial repair was assessed in a scratch assay using vital fluorescence microscopy and compared with a monolayer of A549 cells. RESULTS: HMPEC cells differentially modulated the response of the A549 cells. Surfactant and SP-A augmented the reepithelialization in the presence of HPMECs, whereas in the absence of HPMECs, surfactant inhibited wound healing and SP-A failed to alter the response. Like SP-A, a structural analogue of its collagenous tail domain augmented the reepithelialization in the model system, whereas an analogue of its head domain did not alter the response. Additionally, we demonstrated that TGF-ß associated with SP-A was able to initiate the Smad-dependent TGF-ß pathway and that both TGF-ß and TGF-ß free SP-A were able to stimulate wound healing in the bilayer model. CONCLUSIONS: These data show that surfactant, SP-A and TGF-ß, influence epithelial repair in vitro and that the microvascular endothelial cells can modulate the response. This indicates that surfactant and SP-A could play a role in alveolar epithelial repair and that the microvascular endothelium may be involved in these processes.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

Early alterations of growth factor patterns in bronchoalveolar lavage fluid from preterm infants developing bronchopulmonary dysplasia.

UNLABELLED: Chronic lung disease of prematurity (bronchopulmonary dysplasia; BPD) is characterized by an arrest in lung development. We hypothesized that early alterations in pulmonary expression of growth factors important for normal lung development would precede development of BPD. Bronchoalveolar lavage fluid (BALF) was obtained from ventilated preterm infants (n = 62) on postnatal d 0, 1, 3, and 7 and analyzed for total phospholipids (PL), VEGF, PDGF-BB, TGF-alpha and -beta1, granulocyte macrophage colony stimulating factor (GM-CSF), and keratinocyte growth factor (KGF). Levels (Ln transformed) were compared between infants developing BPD and BPD-free survivors, adjusted for potential confounders. BPD was associated with higher overall GM-CSF (beta (95% CI) = 0.69 (0.13;1.25); p < 0.05), lower overall latent TGF-beta1 (beta (95% CI) = -1.19 (-1.87, -0.39); p < 0.01) and total PL (beta (95% CI) = -0.64 (-1.23, -0.05); p < 0.05), and lower d 0 and 3 levels of VEGF (mean difference (95% CI) = -1.75 (-2.72, -0.77), p < 0.001; and -1.18 (-2.30, -0.06), p < 0.05, respectively) and TGF-alpha (mean difference (95% CI) = -0.73 (-1.42, -0.04), p < 0.05; and -1.01 (-1.64, -0.38), p < 0.01, respectively). Day 0 VEGF levels had the highest predictive value for BPD (area under receiver operating characteristic curve = 0.87; p < 0.01). In conclusion, substantial alterations in BALF growth factor levels are present in infants developing BPD. An early imbalance in pulmonary growth factors may contribute to the developmental arrest of the lung seen in BPD. ABBREVIATIONS: :

Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair.

BACKGROUND: Preterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury in vitro, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised. METHODS: BALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (>or= 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model. RESULTS: On day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 +/- 35.5 vs. -6.3 +/- 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 +/- 87.1 vs. 40.2 +/- 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1. CONCLUSION: Antenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair in vitro. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Protection of the Ovine Fetal Gut against Ureaplasma-Induced Chorioamnionitis: A Potential Role for Plant Sterols.

Chorioamnionitis, clinically most frequently associated with Ureaplasma, is linked to intestinal inflammation and subsequent gut injury. No treatment is available to prevent chorioamnionitis-driven adverse intestinal outcomes. Evidence is increasing that plant sterols possess immune-modulatory properties. Therefore, we investigated the potential therapeutic effects of plant sterols in lambs intra-amniotically (IA) exposed to Ureaplasma. Fetal lambs were IA exposed to Ureaplasma parvum (U. parvum, UP) for six days from 127 d-133 d of gestational age (GA). The plant sterols ß-sitosterol and campesterol, dissolved with ß-cyclodextrin (carrier), were given IA every two days from 122 d-131 d GA. Fetal circulatory cytokine levels, gut inflammation, intestinal injury, enterocyte maturation, and mucosal phospholipid and bile acid profiles were measured at 133 d GA (term 150 d). IA plant sterol administration blocked a fetal inflammatory response syndrome. Plant sterols reduced intestinal accumulation of proinflammatory phospholipids and tended to prevent mucosal myeloperoxidase-positive (MPO) cell influx, indicating an inhibition of gut inflammation. IA administration of plant sterols and carrier diminished intestinal mucosal damage, stimulated maturation of the immature epithelium, and partially prevented U. parvum-driven reduction of mucosal bile acids. In conclusion, we show that ß-sitosterol and campesterol administration protected the fetus against adverse gut outcomes following UP-driven chorioamnionitis by preventing intestinal and systemic inflammation.

Antiviral activity of HIV type 1 protease inhibitors nelfinavir and indinavir in vivo is not influenced by P-glycoprotein activity on CD4+ T cells.

P-glycoprotein (P-gp) can compromise the antiretroviral effect of a protease inhibitor (PI)-containing regimen for HIV-1, but can also reduce HIV-1 replication. We studied the net effect of P-gp on the intracellular HIV-1 RNA and DNA load in vivo. CD4(+) T cells were isolated from 27 HIV-1 patients (13 without and 14 with a PI-containing regimen) and subsequently sorted in CD45RO(-) (naive) and CD45RO(+) (memory) subsets with either high (P-gp(high)) or low (P-gp(low)) P-gp activity. Unspliced HIV-1 RNA and HIV-1 DNA load were determined. For each patient P-gp(high) and P-gp(low) subsets were compared. In patients on a PI-containing regimen, intracellular unspliced HIV-1 RNA was significantly lower in P-gp(high)-naive CD4(+) cells compared to P-gp(low)-naive CD4(+) cells (p = 0.04). The same trend was seen in naive CD4(+) cells of treatment naive patients. In both treated and untreated patients HIV-1 DNA levels were significantly lower in P-gp(high) than in P-gp(low) memory CD4(+) cells (p = 0.02 and p = 0.04). High cellular P-gp activity coincided with a reduced intracellular HIV-1 load in vivo, both in therapy-naive and in PI-treated patients. Therefore we conclude that the potential efflux function of P-gp on PIs may be clinically less relevant than the effect of P-gp on intracellular HIV-1 replication.

Prophylactic Interleukin-2 Treatment Prevents Fetal Gut Inflammation and Injury in an Ovine Model of Chorioamnionitis.

BACKGROUND: Chorioamnionitis results from an infection of the fetal membranes and is associated with fetal adverse outcomes notably in the intestine. Using a translational ovine model, we showed that intra-amniotic exposure to inflammatory stimuli decreased the regulatory/effector T (Treg/Teff) cell balance in the gut, which was accompanied by intestinal inflammation and mucosal injury. We thus aimed to augment the Treg/Teff cell ratio in the fetal gut by prophylactic IL-2 treatment and evaluate whether it is sufficient to prevent chorioamnionitis-induced intestinal inflammation and mucosal injury. METHODS: Fetal sheep (122 d of gestation) were intra-amniotically exposed to lipopolysaccharide for 2 or 7 days with or without prophylactic IL-2 treatment (4 d). We evaluated the infiltration of inflammatory cells in the ileum and mesenteric lymph nodes. Cytokine gene expression was analyzed in fetal ileum and the inflammatory changes were correlated with gut wall integrity. RESULTS: IL-2 administration preferentially increased intestinal Treg cells and thus the Treg/Teff cell ratio. Prophylactic IL-2 treatment reduced the lipopolysaccharide-induced influx of neutrophils and CD3(+) T cells and decreased the messenger RNA levels of proinflammatory cytokines including IL-6 and IL-17 in the fetal ileum. Importantly, prophylactic IL-2 treatment prevented mucosal damage without inducing fetal adverse treatment outcomes. CONCLUSIONS: Our data show that prophylactic IL-2 treatment prevents fetal intestinal inflammation and mucosal injury in the context of experimental chorioamnionitis. Modulation of the Treg/Teff cell balance may contribute to the protective effects of IL-2.

Surfactant protein A binds TGF-ß1 with high affinity and stimulates the TGF-ß pathway.

We were able to demonstrate reversible, specific and high-affinity binding of radioactively-labelled TGF-ß1 ((125)I-TGF-ß1) to immobilized surfactant protein A (SP-A), with an apparent dissociation constant of 53 picomolar at ∼21. Addition of a 200-fold molar excess of the latency associated peptide (LAP) prevented and dissociated the binding of (125)I-TGF-ß1 to SP-A, whereas latent TGF-ß1 had no effect. Using a bioassay for TGF-ß1 activity--a luciferase reporter assay--we were able to show that SP-A in the presence of TGF-ß1 stimulated the TGF-ß1 pathway, whereas SP-A alone had no effect. Studies with structural analogues of the distinct SP-A tail domain and head domain indicated that stimulatory activity of SP-A resided in the head domain. No activation of latent TGF-ß1 by SP-A was observed. In addition, we observed that SP-A inhibited TGF-ß1 inactivation by LAP. These results indicate that SP-A may have a regulatory role in the TGF-ß1-mediated processes in the lung.

Dissociation of transforming growth factors ß1 and ß2 from surfactant protein A (SP-A) by deglycosylation or deoxycholate treatment.

We were able to demonstrate the presence of transforming growth factor ß1 and transforming growth factor ß2 (TGF-ß1,2) in human as well as porcine pulmonary surfactants and SP-A purified from these surfactants. Human SP-A contained 480±74 pg TGF-ß1 and 61±16 pg TGF-ß2 per mg SP-A and human pulmonary surfactant contained 140±28 pg TGF-ß1 and 67±13 TGF-ß2 per mg protein. Porcine SP-A contained 306±46 pg TGF-ß1 and 43±12 pg TGF-ß2 per mg SP-A and porcine pulmonary surfactant contained 75±18 pg TGF-ß1 and 22±13 TGF-ß2 per mg protein. Size-exclusion chromatography indicated binding of TGF-ß1,2 to SP-A. Deglycosylation of SP-A released TGF-ß1,2 from SP-A indicating a role for the carbohydrate moieties of SP-A in binding of TGF-ß1,2. TGF-ß-free SP-A was obtained by incubating SP-A with 5 mM deoxycholate at pH 9.2 followed by size-exclusion chromatography, a protocol which can be used to study the biological activities of SP-A and TGF-ß1,2 separately. In addition, we demonstrated that after incubation of SP-A with TGF-ß1,2, only a part of the added TGF-ß1,2 can be measured, whereas after acid treatment almost all added TGF-ß1,2 was determined, suggesting that complex formation between SP-A and TGF-ß1,2 influences the measurements of TGF-ß1,2 in biological samples.

Pulmonary vascular endothelial growth factor expression and disaturated phospholipid content in a chicken model of hypoxia-induced fetal growth restriction.

BACKGROUND: Prenatal hypoxia is an important cause of intrauterine growth retardation that affects fetal lung maturation, although previous studies have rendered conflicting results. The fetal chicken model allows the study of the isolated effects of hypoxia during development. OBJECTIVES: We hypothesized that prenatal hypoxia would differentially affect surfactant synthesis, depending on timing and duration of hypoxia. Pulmonary vascular endothelial growth factor (VEGF) expression was analyzed as a possible link between oxygen sensing and surfactant production. METHODS: Fertilized White Leghorn eggs were incubated in normoxia, hyperoxia (60% O(2)) from day 15 or hypoxia (15% O(2)) from either day 6 or day 15 of incubation. Whole lung disaturated phospholipids (DSPL) and mRNA expression of VEGF isoforms were quantified at day 16 and 19. RESULTS: Lung DSPL content increased approximately threefold between day 16 and 19 in control animals. Both hypoxia and hyperoxia from day 15 significantly increased DSPL content at day 19 versus control (103 +/- 22 and 116 +/- 18 vs. 81 +/- 15 microg/mg protein, p < 0.01 and p < 0.001, respectively), while long-term hypoxia tended to decrease DSPL content (65 +/- 17 microg/mg protein, p = 0.056). No differences in DSPL content were observed at day 16. Short-term hypoxia transiently up-regulated VEGF146 1.5-fold at day 16 (p < 0.05). A similar trend was observed for VEGF122 (p = 0.058) and VEGF190 (p = 0.08), while no differences were present at day 19. CONCLUSIONS: Both prenatal hypoxia and hyperoxia induced during critical windows of lung development differentially modulate surfactant synthesis. Our data support the concept that fetal oxygen tension is a key signal in the regulation of the surfactant system.

Maternal betamethasone and chorioamnionitis induce different collagenases during lung maturation in fetal sheep.

BACKGROUND: Fetal lung maturation occurs after both maternal corticosteroid administration and chorioamnionitis. The effectors of this antenatally-induced lung maturation are not understood. Matrix metalloproteinases (MMPs) 2 and 9 are type-IV collagenases that can degrade alveolar basement membranes. OBJECTIVES: We hypothesized that the structural changes of lung maturation by both antenatal corticosteroid treatment and chorioamnionitis would be associated with increases in these MMPs. METHODS: 64 pregnant ewes were randomly assigned to one of four treatment groups: intra-amniotic injection of 10 mg endotoxin, maternal intramuscular injection of 0.5 mg/kg betamethasone, both treatments combined or saline-treated controls. We quantified MMP-2 which is derived from connective tissue and MMP-9 which is predominantly derived from neutrophils in fetal lung fluid of lambs after maternal corticosteroid therapy and induction of chorioamnionitis and the combination of both therapies given at 109-111 days' gestational age with delivery 1, 5 or 15 days later. RESULTS: Betamethasone, endotoxin and the combined treatments increased both surfactant pool size, lung gas volume and reduced alveolar wall thickness at 15 days. MMP-2 concentration was increased after betamethasone. MMP-9 concentration increased after endotoxin-induced chorioamnionitis but decreased by the combined treatments. CONCLUSION: Lung maturation via different pathways may use different forms of collagenases for remodelling lung structure.

GB virus C coinfection and HIV-1 disease progression: The Amsterdam Cohort Study.

BACKGROUND: The effect that GB virus C (GBV-C) coinfection has on human immunodeficiency virus type 1 (HIV-1) disease progression is controversial and therefore was studied in 326 homosexual men from the prospective Amsterdam Cohort Studies who had an accurately estimated date of HIV-1 seroconversion and were followed up for a median period of 8 years. METHODS: A first plasma sample, obtained shortly after HIV-1 seroconversion, and a last plasma sample, obtained before 1996, were tested for GBV-C RNA and envelope protein-2 antibodies. The effect that GBV-C has on HIV-1 disease progression was studied by use of time-dependent Cox proportional-hazards models with adjustment for baseline variables and time-updated HIV-1 RNA and CD4(+) cell count. RESULTS: Men who lost GBV-C RNA between collection of the first sample and collection of the last sample had a nearly 3-fold-higher risk of HIV-1 disease progression than did men who had never had GBV-C RNA. This effect became much smaller after adjustment for time-updated CD4(+) cell count. CONCLUSION: Rather than a positive effect of GBV-C RNA presence, a negative effect of GBV-C RNA loss on HIV-1 disease progression was found, which disappeared after adjustment for time-updated CD4(+) cell count. We therefore hypothesize that GBV-C RNA persistence depends on the presence of a sufficient number of CD4(+) cells--and that the CD4(+) cell decrease associated with HIV-1 disease progression is a cause, not a consequence, of GBV-C RNA loss.

Natural controlled HIV infection: preserved HIV-specific immunity despite undetectable replication competent virus.

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naïve individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10(6) PBMC. HIV could not be isolated using up to 30x10(6) patient PBMC. One individual was heterozygous for CCR5 Delta32, but CCR5 expression on CD4+ T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4+ T helper cells were demonstrated by proliferation of CD4+ T cells and intracellular staining for IL-2 and IFNgamma after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection.