IntroductionIdiopathic pulmonary fibrosis (IPF) is a fatal disease of lung parenchymal scarring triggered by alveolar epithelial cell dysfunction. Inherited forms of pulmonary fibrosis, including ...those caused by mutant forms of surfactant protein C (SFTPC), offer an opportunity to study early pathogenic events which remain poorly understood.ObjectivesWe wished to interrogate specific factors involved in SFTPC trafficking to understand the pathological mistrafficking phenotype caused by the commonest pathogenic mutant, SFTPC-I73T, which aberrantly localises to the plasma membrane.MethodsCRISPR-Cas9 forward genetic screens were employed in an immortalised cell model system to interrogate SFTPC processing and trafficking and a FACS-based readout used to identify altered SFTPC localisation. Alveolar organoids derived from human embryonic lung tissue were transduced with an inducible CRISPRi system for the validation of screen hits.ResultsWe identified several novel targets including candidate proteases and ubiquitin ligases in addition to validating core complexes suspected to be involved in SFTPC trafficking. Of particular note is the E3 ligase ITCH which, when depleted, altered SFTPC localisation in a manner that phenocopies the pathogenic I73T mutant. Further validation is underway using genetic manipulation of a novel human embryonic alveolar organoid system.Abstract S108 Figure 1ITCH depletion results in redistribution of SFTPC to the plasma membrane. (A) Subcellular localisation of GFP-SFTPC WT and I73T expressed in HeLa cells. (B) GFP-SFTPC localises at the plasma membrane in ITCH knockout HeLa cellsConclusionsA cell line model of SFTPC trafficking has allowed us to identify important factors in SFTPC trafficking using forward genetic screens which we can test in a physiological system to understand SFTPC handling in health and disease.
Introduction and ObjectivesOur understanding of the pathogenesis of interstitial pulmonary disease is limited largely to its late, fibrotic stages. Inherited forms offer the potential to understand ...earlier pathogenic events. Autosomal dominant mutations in surfactant protein C (SFTPC) cause some cases of familial pulmonary fibrosis; the most common mutation is I73T, which is said to be mistrafficked within the type 2 pneumocyte. We hypothesise that mistrafficking of SFTPC I73T is causal in triggering type 2 pneumocyte dysfunction in familial pulmonary fibrosis. This work aims to understand the mechanism of SFTPC I73T mistrafficking such that type 2 pneumocyte dysfunction in familial, and sporadic disease can be better understood.MethodsWe overexpressed GFP tagged wild-type (WT) and I73T SFTPC in HeLa cells to study their localisation and trafficking. Subcellular localisation and co-localisation with organelle markers was assessed using confocal microscopy and immunofluorescence. Western blotting was used to assess expression of SFTPC isoforms and GFP traps to study SFTPC interactors, including ubiquitin. Mass spectrometry was used to assess the interactome of WT and I73T SFTPC.ResultsIn HeLa cells overexpressing GFP-SFTPC, we observed mistrafficking of SFTPC I73T; WT SFTPC is trafficked to the multivesicular body (MVB), while I73T mistrafficks to the cell surface and into tubular structures (figure 1A) which co-localise with a marker of recycling endosomes Rab8 (figure 1B). Correct trafficking of WT SFTPC to the MVB depends on ubiquitination of lysine-6 (K6). We see an absence of ubiquitination in the I73T mutant and a subcellular distribution similar to that seen in a SFTPC mutant (K6R) which cannot be ubiquitinated. Mass spectrometry revealed differential binding between WT and I73T SFTPC of a number of cell surface proteins.ConclusionWe have shown that pathogenic SFTPC mutant I73T mistrafficks not only to the cell surface, but also into recycling endosomes. We propose that this occurs as a result of failure of ubiquitination and internalisation into the MVB. Trafficking defects appear to impact upon binding of WT vs I73T to cell surface markers, the functional significance of which is the focus of our ongoing work.Abstract T6 Figure 1SFTPC I73T localises to the cell surface and recycling endosomes. (A) Subceller localisation of GFP-SFTPC WT and I73T expressed in HeLa cells. (B) GFP-SFTPC co-localises with mstrawberry Rab8, a marker of recycling endosomes.
A study was conducted to determine effects of bird age at slaughter, feed withdrawal, and transportation on levels of coliforms, Campylobacter, Escherichia coli, and Salmonella on carcasses before ...and after immersion chilling. Broilers were processed at 42, 49, and 56 d of age after a 12-h feed withdrawal period or a 0-h feed withdrawal period (full fed). At each age, broilers were processed from two commercial farms previously identified as Campylobacter positive. One week before slaughter, broilers were gavaged with nalidixic acid-resistant Salmonella. During bleeding, cotton plugs were inserted into the cloaca of each carcass. Whole-carcass rinses (WCR) were performed before and after immersion chilling with 20 ppm sodium hypochlorite, and rinses were analyzed for coliforms, Campylobacter, E. coli and Salmonella. Log10 counts for coliforms, Campylobacter, and E. coli were (P < 0.05) affected by bird age at slaughter. Feed withdrawal (FW) affected only Campylobacter on carcasses of older broilers (56 d of age). Chilling with sodium hypochlorite resulted in log10 reductions of 1.2, 1.3, 1.4, and 0.5 for coliforms, Campylobacter, E. coli, and Salmonella, respectively. Under the conditions of this experiment, it appears that contamination on the exterior of birds entering the processing facility is critical to carcass bacterial counts. Moreover, carcass bacterial counts did not vary when microbial counts of broilers were comparable. FW may increase prechill carcass counts for E. coli and Campylobacter, but it appears to have no effect on postchill carcass counts when sodium hypochlorite is used in the chilling operation.
Alpha-1-antitrypsin is the most abundant circulating protease inhibitor. It is mainly produced by the liver and secreted into the circulation where it acts to prevent excessive proteolytic damage in ...the lungs by the enzyme neutrophil elastase. The most common severe deficiency allele is the Z mutation, which causes the protein to self-associate into ordered polymers. These polymers accumulate within hepatocytes to cause liver damage. The resulting lack of circulating α(1)-antitrypsin predisposes the Z homozygote to proteolytic lung damage and emphysema. Other pathways may also contribute to the development of lung disease. In particular, polymers of Z α(1)-antitrypsin can form within the lung where they act as a pro-inflammatory stimulus that may exacerbate protease-mediated lung damage. Researchers recognized in the 1980s that plasma α(1)-antitrypsin levels could be restored by intravenous infusions of purified human protein. Alpha-1-antitrypsin replacement therapy was introduced in 1987 but subsequent clinical trials have produced conflicting results, and to date there remains no widely accepted clinical evidence of the efficacy of α(1)-antitrypsin replacement therapy. This review addresses our current understanding of disease pathogenesis in α(1)-antitrypsin deficiency and questions why this treatment in isolation may not be effective. In particular it discusses the possible role of α(1)-antitrypsin polymers in exacerbating intrapulmonary inflammation and attenuating the efficacy of α(1)-antitrypsin replacement therapy.
Introduction and objectivesAlpha-1-antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene ...mutation yields Z-alpha-1-antitrypsin, which has a propensity to self-associate into polymers that become entrapped within inclusions of endoplasmic reticulum (ER). This predisposes to the development of cirrhosis, while the resulting paucity of circulating alpha-1-antitrypsin leads to early-onset emphysema. It is unclear whether intracellular inclusions are physically or functionally connected to the main ER network in Z-alpha-1-antitrypsin expressing cells. In this study, we sought to clarify the behaviour of proteins within inclusion bodies to further our understanding of the consequences of Z-alpha-1-antitrypsin expression on cellular dysfunction.MethodsWe created YFP tagged wild-type and Z-alpha-1-antitrypsin constructs and expressed them in a cell model along with other fluorescently tagged proteins of interest. Using live-cell imaging including photobleaching techniques, we assessed the relative mobilities of alpha-1-antitrypsin and other ER resident proteins. We further assessed the nature of inter-inclusion protein trafficking by creating a permeabilised cell system in which the cytosol could be manipulated or removed.ResultsWe have shown that inclusions are translationally active ER fragments in which polymerisation occurs. Using fluorescence recovery after photobleaching (FRAP), we observed that despite inclusions containing immobile polymeric alpha-1-antitrypsin, small ER resident proteins including the ER chaperone BiP are able to diffuse freely within them (Figure 1). We observed that inclusions are physically separated from the tubular ER network but despite this, cargo is transported between inclusions in a cytosol-dependent fashion that is dependent on vesicular trafficking components and may involve the ER-Golgi intermediate compartment (ERGIC).Abstract S126 Figure 1Assessing ER chaperone mobility in cells expressing wild-type and polymerogenic AAT using 2-colour FRAP. Cells expressing WT (A) or polymerogenic Z (B) AAT were subjected to 2-colour FRAP. Note the ER protein ER-mCherry remains mobile even in the presence of immobile Z-AAT. (C) 2-colour bleaching of a single ER inclusion containing Z-AAT confirms immobility of Z-AAT but free movement of ER-mCherry through the immobile AAT lattice. (AAT; al-antitrypsin)ConclusionsWe propose that protein movement between physically separated ER inclusions via ER-ERGIC recycling acts to minimise ER heterogeneity in Z-alpha-1-antitrypsin expressing cells. This may reduce the toxic effects of polymer accumulation via increased availability of, for example, ER chaperones within inclusions and is consistent with in vivo observations of a striking lack of toxicity in cells expressing Z-alpha-1-antitrypsin.
Introduction and Objectives The severe Z deficiency allele of alpha-1-antitrypsin (Glu342Lys) results in the formation of polymers that are retained within hepatocytes, leading to hepatitis, ...cirrhosis and hepatocellular carcinoma. The concomitant lack of circulating protein predisposes to early onset emphysema. Polymers are also found in the lung, skin and kidney and are known to be proinflammatory, but it is unknown whether those polymers are produced locally or are deposited from a circulating source. We wished to establish whether polymers are present in the plasma of individuals with alpha-1-antitrypsin deficiency, from where they originate and whether they are associated with any clinical phenotype. Methods We used a novel anti-alpha-1-antitrypsin polymer monoclonal antibody (2C1) in an ELISA assay to evaluate whether polymers are present in a cohort of 513 individuals with ZZ alpha-1-antitrypsin deficiency. Serial samples from an individual with ZZ alpha-1-antitrypsin deficiency undergoing liver transplantation were used to investigate the source of circulating polymers. We then used a 2nd cohort of 293 individuals with mixed alpha-1-antitrypsin phenotypes to determine whether circulating polymers could be used as a screening test for the presence of a polymerogenic allele. Disease associations were sought using clinical data from the ZZ alpha-1-antitrypsin deficient cohort. Results In the cohort of 513 individuals with PiZZ alpha-1-antitrypsin deficiency, we found 512 had quantifiable polymers present within serum, the 513th having previously had a liver transplant. Circulating polymer levels were higher in men and individuals with COPD and a there was a correlation with older age and lower lung function. The presence of circulating polymers was 100% sensitive and 89% specific in identifying 20 PiZZ alpha-1-antitrypsin homozygotes in a mix of 293 alpha-1-antitrypsin genotypes. Serial blood samples from a PiZZ individual undergoing liver transplantation showed that circulating polymers originate from the liver, clearing with a half-life of approximately 30 hours and becoming undetectable 5 days after transplantation. Conclusions We have shown that circulating polymers are present in PiZZ alpha-1-antitrypsin individuals and originate from the liver. Polymer levels are associated with COPD, suggesting they may play a role in disease pathogenesis. Abstract S64 Figure 1. Z α1 antitrypsin polymers are present in the circulation and are cleared following liver transplantation. Mouse monoclonal antibodies that detect all conformers of α1-antitrypsin, or only polymeric (α1-antitrypsin (2C1), were used to quantify total (α1-antitrypsin and (α1-antitrypsin polymers respectively using sandwich ELISA. Time points marked * are below the lower limit of quantification (0.4 ug/mL).
Protecta II, an herbal extract on an NaCl carrier, was evaluated in a 30-min, 1 C simulated chill for its effectiveness of lowering microbial counts on broiler carcasses. Eighteen broiler carcasses ...were obtained from a local processing plant after final wash but before chill, placed into an insulated container, and transported to the research facility for treatment. Six plant run controls (PRC) were immediately bagged on return to the pilot plant, and a whole-carcass rinse was performed. The remaining carcasses were subjected to a 30-min chill (1 C) in tap water or a 2% solution of Protecta II, (n = 6 per treatment). After treatment, carcasses were rinsed with tap water and subjected to the whole-carcass rinse procedure. All rinse diluents were microbiologically analyzed for total aerobes, coliforms, generic Escherichia coli, and Campylobacter. Six replications were analyzed on 6 different d for a total 36 carcasses per treatment and 36 PRC. The PRC carcasses had 3.7, 2.5, 2.1, and 2.0 log10 cfu/mL for total aerobes, coliforms, generic E. coli, and Campylobacter. Water treatment significantly reduced counts (2.6, 1.4, 0.7, and 0.9 log10 cfu/mL, respectively) when compared with the PRC. Protecta II treatment significantly reduced counts (P < 0.01) even further to counts of 0.06, 0.04, 0.01, and 0.00 log10 cfu/mL for total aerobes, coliforms, Campylobacter, and E. coli, respectively. Detectable levels of the monitored organisms were 1 cell/mL (log10 0) for the E. coli, coliforms, and total counts and 10 cells/mL (log10 1) for the Campylobacter. Microbial counts for carcasses treated with Protecta II would be considered too low to be detected (<1 cell/mL).
Scalding has been found to lower the levels of Campylobacter on broiler carcasses. However, the numbers recovered from whole-carcass rinse samples increase following defeathering. This study was ...undertaken to examine the effect of a second scald applied after defeathering on microbial levels recovered from carcass rinses. Four treatments were evaluated: 1) immersion at 60 C for 28 s 30 min after defeathering, 2) immersion at 60 C for 28 s immediately after defeathering, 3) spray at 73 C for 20 s 30 min after defeathering, and 4) spray at 71 C for 20 s immediately after defeathering. As reported earlier, a significant increase in Campylobacter counts per mL whole carcass rinse was noted after carcasses were defeathered. However, when applied 30 min after defeathering, neither the immersion nor the spray second scald treatments lowered the Campylobacter counts. Likewise, neither treatment had any affect on Escherichia coli or coliform bacteria counts, even though total counts were slightly reduced by the treatments. When the second scald treatment immediately followed defeathering, the same trends were observed. Campylobacter counts after the second scald remained at the postpick levels, as did counts for E. coli and coliform bacteria, but total plate counts were slightly reduced. Overall, it would appear that a postscald treatment gentle enough not to alter the carcass appearance or meat quality would not effectively lower Campylobacter, E. coli, or coliform bacteria counts.
Numbers of Campylobacter detected on broiler carcasses decrease due to scalding but rebound during defeathering. Earlier research indicated that escape of gut contents during defeathering is an ...important contributor to the increase in Campylobacter numbers, whereas aerosols created by feather picking machines are not. The objective of this study was to determine if contact with surfaces in naturally contaminated commercial feather picking machines contributes to the increased counts of Campylobacter on broiler carcasses. Fully processed chilled carcasses with low numbers of Campylobacter were used as a platform to measure numbers after picking. Chilled carcasses were hung on shackles and allowed to proceed through 3 empty feather picking machines in a commercial processing plant that had been previously soiled by about 20,000 broilers. In 4 of 5 replications, although Campylobacter numbers increased on plant run control birds during defeathering, no increase in Campylobacter numbers was noted on chilled carcasses due to passage through the pickers. In one replication, however, a significant increase was observed. It is possible for contact with soiled surfaces within feather picking machines to increase Campylobacter numbers detected on broiler carcasses. However, overall these data suggest that naturally contaminated surfaces of feather picking machines are not a primary cause of the reported increase in Campylobacter numbers during defeathering.
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