The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes

BackgroundNew generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes.MethodsPurified neonatal CD14⁺ cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1β, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry.ResultsCHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1β. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected.ConclusionOur data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1β cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.     

Factors influencing the efficiency of a marker-assisted introgression programme in Merino sheep

This study investigated a marker-assisted introgression programme in Australian Merino sheep. The goal was to introgress an allele with a large negative effect on fibre diameter into a Merino flock possessing medium average fibre diameter. The influence of two factors was explored: the strategy used to select animals from the purebred and backcross line for backcrossing purposes and the use of selection on background markers to accelerate the return to the purebred line''s genome. The results were compared to introgression based on EBVs only. Introgression using EBVs only produced almost the same response in the dollar index as marker-based introgression methods. However, this study did not account for some of the costs associated with implementing the programmes, including the costs of phenotyping and genotyping. Given that the cost of measuring fibre diameter is low, it was concluded that introgression on EBVs only would be the preferred method since the marginal profit of marker-assisted introgression would not be large enough to cover the additional cost of genotyping. In marker-assisted introgression, reciprocal crossing of male and female selection candidates from the backcross and the purebred line was the most advantageous strategy from a practical and profit point of view. Selection for background markers was less profitable in this study than recovering the donor genome by selection on phenotype.     

Robust prediction of mortality of COVID-19 patients based on quantitative,operator-independent,lung CT densitometry

PurposeTo train and validate a predictive model of mortality for hospitalized COVID-19 patients based on lung densitometry.MethodsTwo-hundred-fifty-one patients with respiratory symptoms underwent CT few days after hospitalization. “Aerated” (AV), “consolidated” (CV) and “intermediate” (IV) lung sub-volumes were quantified by an operator-independent method based on individual HU maximum gradient recognition. AV, CV, IV, CV/AV, IV/AV, and HU of the first peak position were extracted. Relevant clinical parameters were prospectively collected. The population was composed by training (n = 166) and validation (n = 85) consecutive cohorts, and backward multi-variate logistic regression was applied on the training group to build a CT_model. Similarly, models including only clinical parameters (CLIN_model) and both CT/clinical parameters (COMB_model) were developed. Model’s performances were assessed by goodness-of-fit (H&L-test), calibration and discrimination. Model’s performances were tested in the validation group.ResultsForty-three patients died (25/18 in training/validation). CT_model included AVmax (i.e. maximum AV between lungs), CV and CV/AE, while CLIN_model included random glycemia, C-reactive protein and biological drugs (protective). Goodness-of-fit and discrimination were similar (H&L:0.70 vs 0.80; AUC:0.80 vs 0.80). COMB_model including AVmax, CV, CV/AE, random glycemia, biological drugs and active cancer, outperformed both models (H&L:0.91; AUC:0.89, 95%CI:0.82–0.93). All models showed good calibration (R²:0.77–0.97). Despite several patient's characteristics were different between training and validation cohorts, performances in the validation cohort confirmed good calibration (R²:0–70-0.81) and discrimination for CT_model/COMB_model (AUC:0.72/0.76), while CLIN_model performed worse (AUC:0.64).ConclusionsFew automatically extracted densitometry parameters with clear functional meaning predicted mortality of COVID-19 patients. Combined with clinical features, the resulting predictive model showed higher discrimination/calibration.     

Decreased surfactant protein B expression in mice derived completely from embryonic stem cells

ES mice that are derived completely from embryonic stem (ES) cells can be obtained by tetraploid embryo complementation. Many neonate ES mice die because of respiratory distress, but it is not clear what contributes to the phenomenon. Using five microsatellite DNA markers, we confirmed that our ES mice were completely derived from ES cells and contained no tetraploid component. The neonatal ES mice that exhibited respiratory distress were tested for surfactant protein B (SP-B) expression by Western blotting. These mice had no SP-B expression, and even apparently healthy adult ES mice had decreased SP-B levels and aberrant SP-B phenotypes. These data suggest that the expression of SP-B protein is an important factor in the survival of ES mice to term and adulthood.     

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

Lung surfactant (LS) is an extra-cellular lipid-protein system responsible for maintaining low surface tension in the lung and alveolar stability. Serum proteins cause dysfunction of this material, e.g. in adult respiratory distress syndrome (ARDS). BLES is a clinically used LS consisting of most of the lipids and associated proteins from bovine lung lavage. Aqueous phases of BLES at 30% and 70% hydration, with and without 5% by weight of bovine serum albumin (BSA), calculated on the amount of lipids, were studied using X-ray diffraction during cooling from 42 to 5 degrees C. The diffraction curves are consistent with a transition from a lamellar liquid crystalline phase to a gel phase transition at cooling in the interval 30-20 degrees C. The long-spacings correspond to a reduction of the bilayer thickness during this transition. The wide-angle region shows a peak at 4.1 A below 25 degrees C, which is characteristic of the hexagonal chain packing of the gel phase. The perturbation of the bilayers by the presence of BSA seems to induce a significant decrease of the bilayer thickness. Calculations on the observed limits of swelling (taking place in the range 50-60%) indicate that BSA is closely associated with the BLES bilayers, probably due to electrostatic interaction with the cationic surfactant proteins SP-B and SP-C. This study show that the LS lipid structural organizations are extremely susceptible to small amounts of serum albumin, which may have implications in surfactant related lung disease and clinical applications of surfactant therapy.     

A critical role of interleukin-1 in preterm labor

Preterm birth (PTB) is a leading cause of neonatal mortality and morbidity worldwide, and represents a heavy economic and social burden. Despite its broad etiology, PTB has been firmly linked to inflammatory processes. Pro-inflammatory cytokines are produced in gestational tissues in response to stressors and can prematurely induce uterine activation, which precedes the onset of preterm labor. Of all cytokines implicated, interleukin (IL)-1 has been largely studied, revealing a central role in preterm labor. However, currently approved IL-1-targeting therapies have failed to show expected efficacy in pre-clinical studies of preterm labor. Herein, we (a) summarize animal and human studies in which IL-1 or IL-1-targeting therapeutics are implicated with preterm labor, (b) focus on novel IL-1-targeting therapies and diagnostic tests, and (c) develop the case for commercialization and translation means to hasten their development.     

Absence of evidence for linkage between Booroola gene and genetic markers at 11 sheep blood polymorphic loci

Summary. Genetic linkage between the Booroola locus ( Fec ) and 11 sheep blood polymorphic loci (i.e. Tf, Hb, CA, OLA, and A, B, C, D, M, R, F41 red cell blood groups) was investigated in six large sire families (163 informative female offspring). The six sires tested were heterozygous for the Booroola allele ( Fec^B ) and for several genetic markers. No evidence in favour of linkage was found. Moreover, depending on the marker locus considered, linkage closer than or as close as the recombination frequency of 10–30% was excluded.     

Synthesis and interfacial behavior of sulfur-containing analogs of lung surfactant dipalmitoyl phosphatidylcholine

Synthesis methods and initial surface property characterizations are reported for two sulfur-containing phosphonolipids related structurally to dipalmitoyl phosphatidylcholine (DPPC), the major lung surfactant glycerophospholipid. Sulfur linkages in these compounds affect molecular interactions relative to ester linkages, and are structurally resistant to cleavage by phospholipases. The SO₂-linked analog synthesized here had increased adsorption and improved film respreading compared to DPPC, while reaching very low surface tensions (1 mN/m) in cycled interfacial films on both the Wilhelmy balance and the pulsating bubble surfactometer. This compound appears to have potential utility as a component in future phospholipase-resistant synthetic exogenous surfactants for treating clinical forms of inflammatory lung injury.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Multilayers at the surface of solutions of exogenous lung surfactant: Direct observation by neutron reflection

Pharmacy-grade exogenous lung surfactant preparations of bovine and porcine origin, dispersed in physiological electrolyte solution have been studied. The organization and dynamics at the air/water interface at physiological temperature was analysed by neutron reflection. The results show that a well-defined surface phase is formed, consisting of a multilayer structure of lipid/protein bilayers alternating with aqueous layers, with a repetition period of about 70 Å and correlation depths of 3 to > 25 bilayers, depending on electrolyte composition and time. The experimental surfactant concentration of 0.15% (w/w) is far below that used in therapeutic application of exogenous surfactants and it is therefore likely that similar multilayer structures are also formed at the alveolar surface in the clinical situation during surfactant substitution therapy. Lung surfactant preparations in dry form swell in aqueous solution towards a limit of about 60% (w/w) of water, forming a lamellar liquid-crystalline phase above about 34 °C, which disperses into lamellar bodies at higher water concentrations. The lamellar spacings in the surface multilayers at the air/water interface are smaller than those in the saturated limit even though they are in contact with much greater water concentrations. The surface multilayers are laterally disordered in a way that is consistent with fragments of Lα-phase lamellae. The near surface layers of the multilayer structure have a significant protein content (only SP-B and SP-C are present in the preparations). The results demonstrate that a multilayer structure can be formed in exogenous surfactant even at very low concentrations and indicate that multilayers need to be incorporated into present interpretations of in vitro studies of similar lung surfactant preparations, which are largely based on monolayer models.     

The growth of sheep scab lesions in relation to sheep breed and time of the year

Numerically equal groups of between five and ten young Merino and Dorper sheep were artificially infested with sheep scab mites, Psoroptes ovis. Separate groups of sheep were infested during the early winter of two successive years as well as during the early summer of the second year. Lesion growth was measured at two-weekly intervals for a period of six weeks during the first winter and for eight weeks during the second winter and in the summer. The rate of lesion growth and mean lesion size on Merino sheep was significantly greater than that on Dorper sheep at each two-weekly recording during the winter of both years. The rate of growth of the lesions did not differ significantly between the two sheep breeds during summer, although, with the exception of eight weeks post-infestation, the mean lesion size on Merino sheep was significantly larger than that on Dorper sheep at each assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of pulmonary surfactant proteins SP-B and SP-C and calcium ions on the surface properties of hydrophobic fractions of lung surfactant

This study focused on two hydrophobic fractions (HF-A and HF-B) isolated from porcine lung surfactant (LS) that had similar phospholipid composition, but HF-A consisted of the hydrophobic LS specific proteins (SP-B and SP-C), in contrast to HF-B. Monolayers spread in a Langmuir trough were formed at the air/water interface of both fractions and the rate of adsorption-desorption and the respreading potential of the LS constituents was studied during six consecutive compression/decompression cycles of the monolayers. By drawing a comparison between the behavior of HF-A and HF-B monolayers on the subphase of 150 mm NaCl, either with or without additional Ca²⁺, we estimated the role of hydrophobic LS proteins and Ca²⁺ ions for LS surface activity. The results demonstrated much higher ability of the HF-A sample, compared to HF-B, to maintain lower surface tension (γ) during monolayer compression and its better respreading capacity during decompression. For instance, at a surface concentration corresponding to 80 Ų per phospholipid molecule, the HF-A monolayers showed a much lower γ _max value (surface tension at 100% of the trough area), being ca. 31.0 mN/m, compared to the HF-B monolayers (γ _max? 62.0 mN/m). The surface tension after compression to 20% of the initial area (γ _min) reached ca. 7.0 and 19.0 mN/m in the HF-A and HF-B monolayers, respectively. Better respreading of the HF-A monolayers compared to the HF-B monolayers was due to the faster adsorption and spreading of LS phospholipids during decompression, facilitated by the hydrophobic proteins. As the phospholipid composition of both fractions was similar, we showed that the hydrophobic surfactant proteins were responsible also for the prevention of the irreversible loss of material from the surface during monolayer compression/decompression. The effects observed demonstrated also that the hydrophobic surfactant proteins were the stronger determinant, compared with Ca²⁺ ions, for the surface tension decrease and respreading of the monolayers during film compression/decompression. For instance, when the HF-A monolayers were spread on a subphase with an additional 5 mm Ca²⁺ ion content, no significant changes were detected in the γ _min and γ _max values between the first and sixth cycle, compared to the monolayers spread on a subphase of 150 mm NaCl only. However, in the absence of positively charged SP-B and SP-C (HF-B sample) in highly compressed monolayers, Ca²⁺ ions were able to cause the effects shown by SP-B and SP-C, although to a less extent. The role of the electrostatic and hydrophobic interactions is discussed for the better respreading of LS components in the presence of LS proteins and Ca²⁺ ions.     

Estimates of (co)variance components and genetic parameters for body weights and first greasy fleece weight in Bharat Merino sheep

(Co)variance components and genetic parameters of weight at birth (BWT), weaning (3WT), 6, 9 and 12 months of age (6WT, 9WT and 12WT, respectively) and first greasy fleece weight (GFW) of Bharat Merino sheep, maintained at Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India, were estimated by restricted maximum likelihood, fitting six animal models with various combinations of direct and maternal effects. Data were collected over a period of 10 years (1998 to 2007). A log-likelihood ratio test was used to select the most appropriate univariate model for each trait, which was subsequently used in bivariate analysis. Heritability estimates for BWT, 3WT, 6WT, 9WT and 12WT and first GFW were 0.05 ± 0.03, 0.04 ± 0.02, 0.00, 0.03 ± 0.03, 0.09 ± 0.05 and 0.05 ± 0.03, respectively. There was no evidence for the maternal genetic effect on the traits under study. Maternal permanent environmental effect contributed 19% for BWT and 6% to 11% from 3WT to 9WT and 11% for first GFW. Maternal permanent environmental effect on the post-3WT was a carryover effect of maternal influences during pre-weaning age. A low rate of genetic progress seems possible in the flock through selection. Direct genetic correlations between body weight traits were positive and ranged from 0.36 between BWT and 6WT to 0.94 between 3WT and 6WT and between 6WT and 12WT. Genetic correlations of 3WT with 6WT, 9WT and 12WT were high and positive (0.94, 0.93 and 0.93, respectively), suggesting that genetic gain in post-3WT will be maintained if selection age is reduced to 3 months. The genetic correlations of GFW with live weights were 0.01, 0.16, 0.18, 0.40 and 0.32 for BWT, 3WT, 6WT, 9WT and 12WT, respectively. Correlations of permanent environmental effects of the dam across different traits were high and positive for all the traits (0.45 to 0.98).     

Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes

Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[²H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D^−/− mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 μmol/lung/h) and SP-D^−/− (0.69 μmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D^−/− mouse was due to impaired catabolism, with a rate of accumulation of 0.057 μmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D^−/− mice before significant PC acyl remodelling occurs.     

Progesterone treatment, FSH plus eCG, GnRH administration, and Day 0 Protocol for MOET programs in sheep

The effect of various superstimulatory treatments on the number of corpora lutea, fertilization rate, and embryo yield was studied in sheep. Overall, data from 708 Merino donors and 4262 embryos were analyzed in four experiments. In Experiment 1, varying intervals of progesterone treatment (5 to 14 d) before follicle-stimulating hormone (FSH) administration did not significantly affect the proportion of responding donors, the mean number of corpora lutea, or the mean number of recovered and transferable embryos per donor. In Experiment 2, a single dose of equine chorionic gonadotropin (eCG, 200 or 300 IU) combined with the FSH treatment (i.e., given at CIDR removal) reduced the number and the quality of embryos compared with that for not giving eCG (P < 0.05). In Experiment 3, one dose of gonadotropin-releasing hormone (GnRH) given 24 h after CIDR removal improved the number of transferable embryos compared with that for not giving GnRH (P < 0.05). In Experiment 4, the new superstimulatory Day 0 Protocol, which includes starting FSH treatment at the emergence of Wave 1 (i.e., soon after ovulation, in the absence of a large follicle), improved ovarian response, with a tendency to produce more embryos compared with that for the Traditional Protocol. In summary, this study, analyzing data from various pharmacologic treatments, allows an improvement from four to eight transferable embryos per treated donor in multiple ovulation and embryo transfer programs in sheep.     

Restoring the charge and surface activity of bovine lung extract surfactants with cationic and anionic polysaccharides

Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air-water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan-lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation.     

大剂量甲泼尼龙救治结缔组织病合并急性呼吸窘迫综合症及文献复习

目的:探讨糖皮质激素在急性呼吸窘迫综合症(ARDS)治疗中的意义及进展。方法:回顾性分析我科采用大剂量甲泼尼龙救治的1例结缔组织病合并ARDS高龄患者诊治过程,并结合文献进行复习。结果:患者采用大剂量甲泼尼龙治疗后,有效改善肺部功能,减轻肺纤维化。随访至今病情稳定。结论:在ARDS治疗过程中,激素发挥重要作用,激素剂量及疗程的应用需因病而异。     

The impact of respiratory gating on lung dosimetry in stereotactic body radiotherapy for lung cancer

The purpose of this study was to evaluate the impacts of respiratory gating and different gating windows (GWs) on lung dosimetry in stereotactic body radiotherapy (SBRT) for lung cancer.Gated SBRT plans were developed using the four-dimensional computed tomography data from 17 lung cancer patients treated with SBRT. Using amplitude-based end-exhalation gating, we established 2 fixed GWs with approximate duty cycles of 50% (50% GW) and 25% (25% GW), respectively, for this study.For highly mobile tumors (3D mobility > 10 mm), additional benefits in lung-dose reductions were achieved with the 25% GW, as a result of inadequate mobility and planning target volume reductions obtained with the 50% GW. In these tumors, the absolute differences compared to the non-gated and 50% gated plans, were 0.5 Gy and 0.33 Gy for the mean lung dose and 1.11% and 0.71% for the V20, respectively. Dosimetric benefits were achieved with the 50% GW, compared with the non-gated plan, for tumors with both low mobility and small volume (gross tumor volume ≤ 10 cc). Among the identified predictive factors of dosimetric benefits, the lateral distance from midspinal canal and the motion range in anterior–posterior direction might be stronger factors because of their correlations with many of the lung-dose parameters and greater predictive capacity.The results of the present study might facilitate the selection of appropriate patients and the optimal GW according to the tumor characteristics for gated lung SBRT.