Paradoxical effects of cigarette smoke and COPD on SARS-CoV-2 infection and disease.

BACKGROUND: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro. METHODS: We quantified: (1) pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and/or TMPRSS2 mRNA levels by RT-qPCR in two independent human cohorts; and (2) pulmonary ACE2 protein levels by immunostaining and ELISA in C57BL/6 WT mice exposed to air or CS for up to 6 months. The effects of CS exposure on SARS-CoV-2 infection were evaluated after in vitro infection of Calu-3 cells and differentiated human bronchial epithelial cells (HBECs), respectively. RESULTS: ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus controls but similar in central airways. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice. CS treatment decreased viral replication in Calu-3 cells, as determined by immunofluorescence staining for replicative double-stranded RNA (dsRNA) and western blot for viral N protein. Acute CS exposure decreased in vitro SARS-CoV-2 replication in HBECs, as determined by plaque assay and RT-qPCR. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-exposure potently inhibited SARS-CoV-2 replication in vitro. These findings urge to investigate further the controversial effects of CS and COPD on SARS-CoV-2 infection.

Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease.

INTRODUCTION: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. METHODS: In Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. RESULTS: In both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.

Analysis of pulmonary non-tuberculous mycobacterial infections after lung transplantation.

PURPOSE: Non-tuberculous mycobacteria (NTM) are important pathogens in lung transplant recipients. This study describes the spectrum of NTM respiratory tract infections and examines the association of NTM infections with lung transplant complications. METHODS: Data from 208 recipients transplanted from November 1990 to November 2005 were analyzed. Follow-up data were available to November 2010. Lung infection was defined by bronchoalveolar lavage, sputum, or blood cultures in the appropriate clinical setting. All identified NTM respiratory tract infections were tabulated. The cohort of patients with NTM lung infections (NTM+) were compared to the cohort without infection (NTM-). Univariate and multivariate analysis was performed to determine characteristics associated with NTM infection. Survival analyses for overall survival and development of bronchiolitis obliterans syndrome (BOS) were also performed. RESULTS: In total, 52 isolates of NTM lung infection were identified in 30 patients. The isolates included Mycobacterium abscessus (46%), Mycobacterium avium complex (MAC) (36%), Mycobacterium gordonae (9%), Mycobacterium chelonae (7%), and Mycobacterium fortuitum (2%), with multiple NTM isolates seen on 3 different occasions. The overall incidence was 14%, whereas cumulative incidences at 1, 3, and 5 years after lung transplantation were 11%, 15%, and 20%, respectively. Comparisons between the NTM+ and NTM- cohorts revealed that NTM+ patients were more likely to be African-American and have cytomegalovirus mismatch. Although no difference was seen in survival, the NTM+ cohort was more likely to develop BOS (80% vs. 58%, P = 0.02). NTM+ infection, however, was not independently associated with development of BOS by multivariate analysis. CONCLUSION: With nearly 20 years of follow-up, 14% of lung recipients develop NTM respiratory tract infections, with M. abscessus and MAC more commonly identified. M. gordonae was considered responsible for nearly 10% of NTM infections. Although survival of patients with NTM infections is similar, a striking difference in BOS rates is present in the NTM+ and NTM- groups.

Probable Phaeoacremonium parasiticum as a cause of cavitary native lung nodules after single lung transplantation.

Lung nodules after lung transplantation most often represent infection or post-transplant lymphoproliferative disorder in the allograft. Conversely, native lung nodules in single lung transplant recipients are more likely to be bronchogenic carcinoma. We present a patient who developed native lung cavitary nodules. Although malignancy was anticipated, evaluation revealed probable Phaeoacremonium parasiticum infection. Phaeoacremonium parasiticum is a dematiaceous fungus first described as a cause of soft tissue infection in a renal transplant patient. Lung nodules have not been previously described and this is the first case, to our knowledge, of P. parasiticum identified after lung transplantation.

Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

Listeria grayi bacteremia in a heart transplant recipient.

Bacteria belonging to the genus Listeria have been isolated from food products of animal, plant, and fish origin, and are associated with infections in immunocompromised hosts, pregnant women, and infants. The species Listeria grayi has rarely been reported as a human pathogen. It has a unique antibiotic sensitivity profile. We describe a case of L. grayi bacteremia in a heart transplant recipient. The organism demonstrated a reduced sensitivity to ampicillin. The patient was successfully treated with a combination of vancomycin and ciprofloxacin.

Extrinsic compression of the left main coronary artery in pulmonary hypertension.

Compression of the left main coronary artery by a dilated pulmonary artery can be a life-threatening condition. It can present with angina, symptoms of left ventricular dysfunction, and sudden death. Diagnosis and treatment of this condition is very important. We demonstrate significant compression of the left main coronary artery on coronary angiography and thorax CT examinations.

Contained myocardial rupture: a variant linking complete and incomplete rupture.

Myocardial rupture is an uncommon complication of myocardial infarction, often with devastating haemodynamic consequences. Although rupture is usually fatal, when patients do survive, the majority present with a pseudoaneurysm in which the rupture is sealed by a haematoma on the epicardial surface of the heart. Cases in which all myocardial layers are dissected except the epicardium or visceral pericardium have been included under this subheading. The authors describe such a case and suggest the pathological description of a "contained myocardial rupture". This link between complete and incomplete myocardial rupture may allow a more conservative management approach to be pursued.

Development of an ambulatory artificial lung in an ovine survival model.

We are developing an artificial lung (AL) as an eventual bridge to lung transplant or recovery. The device is rigidly housed, noncompliant, and has a very low resistance to blood flow. In eight sheep, arterial cannulae were anastomosed end-to-side to the proximal and distal main pulmonary artery, and attached to the AL. A pulmonary artery snare between anastomoses diverted full pulmonary blood flow through the AL. Eight of eight sheep survived the preparation. Mean pressure gradient across the AL was 8 mm Hg (3 Wood units; 8 mm Hg/2.8 L/min). Four of eight sheep tolerated immediate full diversion of blood flow and died at 24 and 40 hours (exsanguination) or 168 and 168 hours (elective sacrifice). Four of eight sheep were intolerant of full flow: two died of right heart failure at <8 hours with full flow through the device (full snare); the other two survived with partial device flow (partial snare), but the device clotted. These two then underwent successful closed-chest cannula thrombectomy and device change-out at 53 and 75 hours, and subsequently tolerated full flow. Long-term (up to 7 day) survival with complete diversion of pulmonary blood flow through a non-compliant, low-resistance AL is possible. Initial right heart failure in this model was 50% (4 of 8).

Improved right heart function with a compliant inflow artificial lung in series with the pulmonary circulation.

BACKGROUND: We previously reported a 50% incidence of immediate right heart failure using a rigidly housed, noncompliant inflow artificial lung in series with the pulmonary circulation in a healthy ovine survival model. Three device modifications resulted: (1) an inflow cannula compliance chamber, (2) an inlet blood flow separator, and (3) modification of the artificial lung outlet geometry, all to reduce resistance and mimic the compliance of the pulmonary vascular bed. METHODS: In 7 sheep, arterial grafts were anastomosed end-to-side to the proximal and distal main pulmonary artery, with the paracorporeal artificial lung interposed. A pulmonary artery snare between anastomoses diverted full pulmonary blood flow through the artificial lung for up to 72 hours. RESULTS: Six of 7 sheep exhibited good cardiac function throughout the test period: mean central venous pressure was 6.8 mm Hg (range, 4 to 11 mm Hg), mean cardiac output, 4.17 +/- 0.12 L/min (range, 2.4 to 6.3 L/min); before and after device mean pulmonary arterial pressure, 21.8 and 18.5 mm Hg, and left atrial pressure, 10.8 mm Hg. CONCLUSIONS: This modified artificial lung prototype with an inflow compliance chamber, blood flow separator, and modified outlet geometry has greatly improved cardiac function and initial survival in our healthy ovine model.

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the terminal ileum of fistulated Göttingen minipigs.

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10(6) to 10(7) per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.

Development of an implantable artificial lung: challenges and progress.

Unlike dialysis, which functions as a bridge to renal transplantation, or a ventricular assist device, which serves as a bridge to cardiac transplantation, no suitable bridge to lung transplantation exists. Our goal is to design and build an ambulatory artificial lung that can be perfused entirely by the right ventricle and completely support the metabolic O2 and CO2 requirements of an adult. Such a device could realize a substantial clinical impact as a bridge to lung transplantation, as a support device immediately post-lung transplant, and as a rescue and/or supplement to mechanical ventilation during the treatment of severe respiratory failure. Research on the artificial lung has focused on the design, mode of attachment to the pulmonary circulation, and intracorporeal versus paracorporeal placement of the device.

Anatomic study of the pulmonary artery as a conduit for an artificial lung.

Our group is developing an artificial lung as a bridge to transplant. We evaluated the sheep pulmonary artery (PA) for the presence or absence of a septum, which may increase PA resistance and affect artificial lung flow. We also measured the PA size to determine whether it is a suitable conduit for artificial lung implantation using a PA-PA shunt. Adult Suffolk ewes in two groups were studied. Group 1 consisted of animals (n = 12, 30-43 kg) prepared for thoracotomy. Group 2 (n = 21, 30-43 kg) consisted of postmortem dissections. In both groups, the length and girth of the PA was measured. The heart and lungs were removed on all postmortem animals (group 2), the ductus arteriosum was crosscut, and the common PA was incised. The average length of the PA in live animals was 5.5 cm and the average diameter was 2.2 cm. The average length of the PA in postmortem animals was 4.8 cm and the average diameter was 2.0 cm. All pulmonary arteries were aseptate, and the ligamentum arteriosum in each PA was not patent. We conclude that the PA is not a source of increased resistance and is a suitable conduit for artificial lung implantation in the PA-PA configuration.

Identification of lactobacillus delbrueckii and subspecies by hybridization probes and PCR.

Three methods addressing two different target sites were compared for identification and differentiation of the subspecies Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis/delbrueckii. A PCR method - three primer pairs that enable direct identification of the species and the two subspecies, respectively - was derived from a DNA fragment showing significant similarities to parts of the addAB genes of Bacillus sutbtilis. In addition, two oligonucleotide probes for the two subspecies were designed from that DNA region. Further, two oligonucleotide probes targeting the 16S rDNA were developed for subspecies differentiation by a one base-pair difference following identification of the species. Moreover, these probes were demonstrated to be applicable for in situ hybridization experiments. The results obtained by the different methods were in good agreement.

Technique of controlled reperfusion of the transplanted lung in humans.

BACKGROUND: Reperfusion injury remains a significant and sometimes fatal problem in clinical lung transplantation. Controlled reperfusion of the transplanted lung using white cell-filtered, nutrient-enriched blood has been shown recently to significantly ameliorate reperfusion damage in a porcine model. We modified this experimental technique and applied it to human lung transplantation. METHODS: Approximately 1,500 mL of arterial blood was slowly collected in a cardiotomy reservoir during the lung implant, and mixed to make a 4:1 solution of blood:modified Buckberg perfusate. This solution was passed through a leukocyte filter and into the transplant pulmonary artery for 10 minutes, at a controlled rate (200 mL/min) and pressure (less than 20 mm Hg), immediately before removal of the vascular clamp. RESULTS: Five patients underwent lung transplantation (1 bilateral, 4 single lung) using this technique. All patients were ventilated on a 40% fraction of inspired oxygen within a few hours and extubated on or before the first postoperative day. CONCLUSIONS: Controlled reperfusion of the transplanted lung with white cell-filtered, nutrient-enriched blood has given excellent functional results in our small initial clinical series.

Sporulation of Penicillium camemberti in submerged batch culture.

Sporulation of Penicillium camemberti was studied in submerged batch fermentation. A defined medium was used with glucose and ammonium as C- and N-sources. Temperature was set to 25 degrees C at pH 5.6. Essential for submerged sporulation was the presence of calcium (14 mM) which was adsorbed to the cell walls in all sporulating strains and inhibited mycelial growth. Acetate led to highly branched conidiophores and was the second main factor for efficient sporulation. The chelating properties of citrate were necessary for keeping calcium and phosphate in solution. Fermentation conditions allowed high spore yields after 96 h (1.6 x 10(8) spores/ml). Cyclopiazonic acid, the mycotoxin common for P. camemberti was produced during fermentation. The levels observed (0.5-4 ppm at 96 h) were strain specific and not related to spore yield.

Thrombolysis with resolution of pulmonary hypertension in a heart transplant candidate.

We report a patient with idiopathic cardiomyopathy and high pulmonary resistance due to pulmonary emboli of unknown age. Successful thrombolytic therapy returned his pulmonary resistance to normal, allowing orthotopic cardiac transplantation. This case underscores the need to aggressively diagnose and treat pulmonary emboli in potential transplant candidates.