The Role of Pulmonary Surfactant Phospholipids in Fibrotic Lung Diseases.

Diffuse parenchymal lung diseases (DPLD) or Interstitial lung diseases (ILD) are a heterogeneous group of lung conditions with common characteristics that can progress to fibrosis. Within this group of pneumonias, idiopathic pulmonary fibrosis (IPF) is considered the most common. This disease has no known cause, is devastating and has no cure. Chronic lesion of alveolar type II (ATII) cells represents a key mechanism for the development of IPF. ATII cells are specialized in the biosynthesis and secretion of pulmonary surfactant (PS), a lipid-protein complex that reduces surface tension and minimizes breathing effort. Some differences in PS composition have been reported between patients with idiopathic pulmonary disease and healthy individuals, especially regarding some specific proteins in the PS; however, few reports have been conducted on the lipid components. This review focuses on the mechanisms by which phospholipids (PLs) could be involved in the development of the fibroproliferative response.

Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice.

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

Organic synthesis of 1,2-dipalmitoyl-rac-glycero-3-phosphatidylethanolamine and its effect on the induction of apoptosis in normal human lung fibroblasts.

BACKGROUND /OBJECTIVE: The phospholipid 1,2-dipalmitoyl-rac-glycero-3-phosphatidylethanolamine (PE) comprises two fatty acid chains: glycerol, phosphate, and ethanolamine. PE participates in critical cellular processes such as apoptosis and autophagy, which places it as a target for designing new therapeutic alternatives in diseases such as pulmonary fibrosis. Therefore, this study aimed obtain PE through a six-step organic synthesis pathway and determine its biological effect on apoptosis induction in normal human lung fibroblasts (NHLF). METHODOLOGY: The first step of the organic synthesis route began with protected glycerol that was benzylated at sn-3; later, it was deprotected to react with palmitic acid at sn-1, sn-2. To remove the benzyl group, hydrogenation was performed with palladium on carbon (Pd/C); subsequently, the molecule was phosphorylated in sn-3 with phosphorus oxychloride and triethylamine, and the intermediate was hydrolyzed in an acid medium to obtain the final compound. After PE synthesis, apoptosis assessment was performed: apoptosis was induced using exposure to annexin V-FITC/propidium iodide-ECD (PI) and quantified using flow cytometry. The experiments were performed in three NHLF cell lines with different concentrations of PE 10, 100 and 1000 µg/mL for 24 and 48 h. RESULTS: The PE obtained by organic synthesis presented a melting point of 190-192 °C, a purity of 95%, and a global yield of 8%. The evaluation of apoptosis with flow cytometry showed that at 24 h, exposure to PE 10, 100, and 1000 µg/mL induces early apoptosis in 19.42%- 25.54%, while late apoptosis was only significant P < 0.05 in cells challenged with 100 µg/mL PE. At 48 h, NHLF exposed to PE 10, 100, and 1000 µg/mL showed decreasing early apoptosis: 28.69-32.16%, 12.59-18.84%, and 10.91-12.61%, respectively. The rest of the NHLF exposed to PE showed late apoptosis: 12.03-16-42%, 11.04-15.94%, and 49.23-51.28%. Statistical analysis showed a significance P < 0.05 compared to the control. CONCLUSION: The organic synthesis route of PE allows obtaining rac-1,2-O-Dipalmitoyl-glycero-3-phosphoethanolamine (1), which showed an apoptotic effect on NHLF.

Y chromosome microdeletions in Mexican males of couples with idiopathic recurrent pregnancy loss.

AIM: To analyze the presence of Y chromosome microdeletions in males of Mexican couples with idiopathic recurrent pregnancy losses (RPL). METHODS: Seventy-one males from couples with RPL and 66 fertile males as controls were studied. DNA was isolated from peripheral lymphocytes and used to run multiplex polymerase chain reactions. Regions AZFa (sY84, sY86), AZFb (sY127, sY134) and AZFc (sY254, sY255) of the Y chromosome were analyzed according to valid guidelines recommended by the European Academy of Andrology and the European Molecular Genetics Quality Network. Also, the sequence tagged sites (STSs): DYS262 (sY67), DYS220 (sY129), DYF85S1 (sY150), DYF86S1 (sY152) and DYF87S1 (sY153) were included in order to analyze STSs previously reported as deleted. A power analysis to support our simple size was performed. RESULTS: Results show an absence of Y chromosome microdeletions in males of couples with RPL and controls with an acceptable statistical power. CONCLUSION: The study did not show an association of recurrent pregnancy loss and Y chromosome microdeletions in Mexican male partners. Based on the results, the study of Y chromosome microdeletions in couples with RPL is not considered clinically relevant.

Absence of association between nailfold capillary findings and mild cognitive dysfunction in systemic lupus erythematosus.

OBJECTIVE: The role of vascular damage in cognitive dysfunction (CD) in SLE is not entirely understood. Nailfold capillaroscopy (NFC) is a noninvasive method that may aid the description of further vascular contributions to CD in SLE. Therefore, the aim of our study was to examine and compare finger nailfold capillary morphology in subjects with SLE with and without CD. METHODS: We conducted a cross-sectional study in patients with SLE. Demographic, clinical, and laboratory characteristics were collected. We evaluated nailfold capillary findings including avascular zones, hemorrhage, dilated and tortuous capillaries, disarrangement, crossing, subpapillary venular plexus, branched loops, and shortened loops by NFC. The Montreal Cognitive Assessment (MoCA) scale was used to screen cognitive function. CD was defined as a score < 26/30. RESULTS: Sixty-five females (97.0%) and 2 males (3%) with SLE were analyzed. Means of age and disease duration were 44.3 ± 12.0 years and 15.5 ± 7.6 years, respectively. Thirty-five (54.7%) patients had CD. The rate of patients with ≥ 1 NFC abnormality was 50% in both patients with and without CD (P = 0.14). Eight (22.8%) patients with CD compared to 1 without (3.5%) displayed dilated capillaries (P = 0.036). Other NFC abnormalities differed between patients with and without CD, but the possible relationships between dilated capillaries and CD disappeared after adjusting by age, diabetes, and hypertension. CONCLUSIONS: NFC findings were not associated with mild CD in patients with SLE. Our exploratory data do not support systemic microvasculopathy measured by NFC related to CD in patients with SLE.

Functional and Aesthetic Osseous Free Flap Mandible Reconstruction Using a Low-cost Method: Long-term Results.

Mandibular segment loss due to tumoral resection or infectious sequelae causes functional alterations with disorders in mastication, swallowing, speech, and aesthetic alterations with facial asymmetry due to deformity secondary to soft tissue collapse. Reconstructive treatment should recover function and aesthetics. This paper presents a low-cost method of mandibular reconstruction in a series of 6 patients with different years of follow-up (average follow-up time of 11.6 years) using the fibula and scapula free flaps. METHODS: Five female patients and 1 male patient received mandibular reconstruction using osseous free flaps, 5 with the fibula and 1 with the scapula osseous free flap. The patient's ages at the time of surgery ranged from 8 to 62 years (mean 33.1 years). Stainless steel wire was used as the osteosynthesis material, with intermaxillary fixation for 40 days postoperatively and masticatory rehabilitation using mucodental-supported prostheses. RESULTS: To evaluate the aesthetic result and the facial symmetry, a questionnaire and the photographs of all the cases were sent to 8 plastic surgeons. The functional result was evaluated in 5 of the 6 patients using the Spanish version of the Oral Health Impact Profile. All flaps survived, dental occlusion was achieved in all patients, no tumors recurred, masticatory function was normal without swallowing or speech alterations, and the transplanted bone hypertrophied and spontaneously remodeled, providing facial symmetry with good aesthetic results. CONCLUSION: We present a low-cost and universally applicable mandibular reconstruction method, with long-term follow-up and good aesthetic and functional results.

[A new laparoscopic simulator]. / Un nuevo simulador de laparoscopia.

BACKGROUND: Simulators are effective devices for the development of certain skills needed in laparoscopic surgery. Bench models with laparoscopy equipment, virtual reality and mirror boxes have been used; however, they have limitations such as the need for laparoscopy equipment, high cost or a considerable mismatch with reality. METHODS: We undertook this study to test a simulator as a training device that allows the acquisition of eye-hand coordination and two-dimensional spatial orientation without the need of laparoscopic equipment. The simulator consists of a box with an internal light and a color video CCD connected to a television set. Quality of vision, illumination and adequacy as training equipment was assessed by experienced laparoscopic surgeons. Thereafter, 12 general surgeons without experience in laparoscopic surgery and 18 surgery trainees performed seven different drills after a short course on basic skills and knot-tying laparoscopic techniques. The time to completion of each task was recorded at the beginning and after 10 practices of 30-min each. RESULTS: Ten experienced surgeons qualified with the simulator with a mean of 42 points (40-44 from a 44 total). There were significant reductions in the final times of all participants. Paired t-test was significant in all the measurements. The mean time decreased 31.8% (from 1108 +/- 96 to 755 +/- 107 sec) with a 95% confidence interval of 15.1-48.5%. CONCLUSIONS: The simulator tested in this study helps to develop laparoscopic surgical skills economically and without the need for laparoscopic equipment. This laparoscopy training equipment is novel and original in its design.

Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts.

Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 µM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 µg/ml. The application of beractant, at a concentration of 500 µg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cß (PLCß), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.

One, two, or three ports in laparoscopic cholecystectomy?

Single-port laparoscopic cholecystectomy (LC) has been compared with 3- or 4-port LC. To our knowledge, there are no studies comparing the 3-, 2-, and 1-port techniques. Patients were randomized into 3 groups: LC 1-port using SILS, LC 2-port using a laparoscope with a working channel, and LC 3-port using the standard ports. Pain was evaluated at recovery, 4 hours, 24 hours, day 5, and day 8, using an analog visual scale. Homogenous groups in their demographic characteristics; all confirmed gallbladder lithiasis. At recovery, there was less pain in group 1 (P = 0.002); at 4 hours pain was similar in all groups (P = 0.899); at 24 hours there was less pain in groups 2 and 3 (P = 0.031); and at days 5 and 8 there was marginal (P = 0.053) and significant (P = 0.003) relevance. In terms of pain perception, LC performed through 1 port does not offer advantages when compared with 2 or 3 ports. More clinical trials are needed to confirm these data.

Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells.

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.