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.

Effect of blood product transfusion-induced tolerance on incidence of cardiac allograft rejection.

BACKGROUND: Blood product transfusion has been successfully used in solid-organ transplantation to induce tolerance. Whether a similar protective effect of blood product transfusion exists in heart transplantation is controversial. OBJECTIVE: To investigate the effect of cellular blood product transfusion within 2 weeks posttransplantation on the incidence of cellular and antibody-mediated rejection. PATIENTS AND METHODS: Patients were grouped on the basis of number of blood transfusions; group 1 received no transfusions, and groups 2, 3, and 4 each received an incremental number of transfusion units. All endomyocardial biopsy samples were routinely studied using immunofluorescence in the first 12 weeks posttransplantation. RESULTS: Baseline characteristics including age, sex, body mass index, history of diabetes, donor characteristics, and pretransplantation laboratory values were similar except that group 4 had a higher rate of previous sternotomy and longer ischemic time during transplantation. Approximately 9200 endomyocardial biopsy samples composed the data. Short- and long-term freedom from the International Society for Heart & Lung Transplantation grade 3A or higher cellular rejection and from antibody-mediated rejection were comparable between groups. CONCLUSIONS: Blood transfusions within the first 2 weeks post-transplantation do not seem to confer any protective effect against posttransplantation cellular rejection or antibody- mediated rejection. Whether other unmeasured confounding factors mask their effect requires further prospective studies.

Polymeric gene delivery of ischemia-inducible VEGF significantly attenuates infarct size and apoptosis following myocardial infarct.

The development of clinically beneficial myocardial gene therapy has been slowed by reliance on the use of viral carriers and non-physiologic, constitutive gene expression. To specifically address these issues, we have developed a non-viral gene carrier, water-soluble lipopolymer (WSLP), and an ischemia-inducible plasmid construct expressing vascular endothelial growth factor (VEGF), pRTP801-VEGF, to treat myocardial ischemia and infarction. Rabbits underwent ligation of the circumflex artery followed by injection of (a) an ischemia-inducible VEGF gene construct in a WSLP carrier; (b) a constitutively expressed, or unregulated, SV-VEGF gene construct in a WSLP carrier; (c) WSLP carrier alone; or (d) no injection therapy. Following 4 weeks treatment, ligation alone resulted in infarction of 48+/-7% of the left ventricle. With injection of WSLP carrier alone, 49+/-6% of the left ventricle was infarcted (P=NS). The constitutively expressed gene construct, SV-VEGF, reduced the infarct size to 32+/-7% of the left ventricle (P=0.007). The ischemia-inducible gene construct, RTP801-VEGF, further reduced the infarct size to 13+/-4% of the left ventricle (P<0.001). The use of a non-viral carrier to deliver an ischemia-inducible VEGF construct is effective in the treatment of acutely ischemic myocardium.

Hypoxia-inducible VEGF gene delivery to ischemic myocardium using water-soluble lipopolymer.

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a new potential treatment in cardiovascular disease. However, unregulated VEGF-mediated angiogenesis has the potential to promote tumor growth, accelerate diabetic proliferative retinopathy, and promote rupture of atherosclerotic plaque. To be safe and effective, gene therapy with VEGF must be regulated. To limit the risk of pathological angiogenesis, we developed a hypoxia-inducible VEGF gene therapy system using the erythropoietin (Epo) enhancer and water-soluble lipopolymer (WSLP). pEpo-SV-VEGF or pSV-VEGF-Epo was constructed by insertion of the Epo enhancer upstream of the Simian Virus 40 (SV40) promoter or downstream of the poly(A) signal of pSV-VEGF. In vitro transfection showed that pEpo-SV-VEGF, not pSV-VEGF-Epo, induced the VEGF expression in hypoxic cells. In addition, the VEGF protein, which was produced from the Epo-SV-VEGF-transfected and hypoxia-incubated cells, was able to enhance the proliferation of the endothelial cells. Injection of the pEpo-SV-VEGF/WSLP complex showed that the expression of VEGF was induced in ischemic myocardium, compared to normal myo-cardium. Therefore, with the localized induction of VEGF and the low cytotoxicity of WSLP, the pEpo-SV-VEGF/WSLP system may be helpful to eventually treat ischemic heart disease.

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium.

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.

Effect of a resident self-study and presentation program on performance on the thoracic surgery in-training examination.

BACKGROUND: In this study we examine whether conversion from a didactic lecture format to a resident self-study and presentation program can improve performance on the Thoracic Surgery In-Training Examination (TSITE). METHODS: During the first 5 years, educational conferences were didactic lectures delivered by the attending thoracic surgery staff (group 1, n = 9 residents). During the second 5 years, residents prepared and delivered reviews from major textbook sources (group 2, n = 9 residents). Scores on the American Board of Surgery In-Training Examination (ABSITE) as a chief resident in general surgery were analyzed using one-way analysis of variance to assess fund of knowledge and test-taking skills prior to thoracic surgery training for the two groups. Scores on the TSITE during the first and second years of thoracic surgery training were recorded for each resident and analyzed using a paired t test. The data are expressed as the mean +/- standard deviation. RESULTS: Eighteen thoracic surgery residents over a 10-year period were involved in the study. ABSITE scores as a chief resident in general surgery did not differ between the two groups. Residents in group 1 improved their percentile rank from the first to the second year by a mean of 11%+/-12%, whereas those in group 2 improved their scores by a mean of 31%+/-21% (P < 0.05). CONCLUSIONS: When compared with a didactic lecture format, a resident self study and presentation program improves performance on the Thoracic Surgery In-Training Examination. This improvement in performance typically manifests during the second year of thoracic surgery training.

Augmentation of myocardial transfection using TerplexDNA: a novel gene delivery system.

Gene therapy is a potential new strategy for the treatment of cardiovascular disease. The most efficacious method of gene delivery remains a key hurdle to effective gene therapy. We present the application of a novel, nonviral gene delivery system (TerplexDNA) to augment myocardial transfection. The hearts of New Zealand white rabbits were injected with reporter genes, luciferase cDNA or beta-galactosidase cDNA, either as naked plasmid DNA or plasmid DNA complexed with stearyl-poly(L-lysine)-low density lipoprotein (TerplexDNA). Three day left heart myocardial cell lysates produced 44571 +/- 8730 RLU (RLU = total light units/mg protein) for the TerplexDNA luciferase rabbits versus 1638 +/- 567 RLU for the naked luciferase rabbits (P = 0.002). Thirty days after injection, myocardial lysates produced 677 +/- 52 RLU for the TerplexDNA luciferase hearts versus 18 +/- 3 RLU for the naked luciferase hearts (P = 0.002). Histologic analysis of the hearts transfected with beta-galactosidase showed that TerplexDNA increased the area and depth of transfection compared with the naked plasmid DNA alone. The hearts of Sprague-Dawley rats were injected in a similar fashion and analyzed at 1, 3, 5, 10, 15, 25 and 30 days after injection. The naked luciferase injected hearts showed transient elevation of luciferase activity to day 5 but fell back to baseline levels after that time-point. The TerplexDNA luciferase injected hearts had significantly elevated luciferase activity to 30 days. The Terplex gene delivery system significantly augments myocardial transfection compared with a naked plasmid DNA system alone. The advantage in transfection efficiency appears to be related to the unique properties of the TerplexDNA carrier molecule. The TerplexDNA delivery system represents a novel means to augment transfection of the myocardium.

Interleukin-18 production following murine cardiac transplantation: correlation with histologic rejection and the induction of INF-gamma.

Interleukin-18 (IL-18) and IL-12 have been shown to play an important role in the induction of interferon-gamma (IFN-gamma). IFN-gamma induces the proliferation of T cells and natural killer (NK) cells and augments the Th1 immune cascade. The role of IL-18 and IL-12 in the induction of IFN-gamma following allogeneic heart transplantation has not been described. We sought to characterize the IL-12 and IL-18 response to murine allogeneic heart transplantation, particularly with respect to IFN-gamma production and histologic transplant rejection. Forty-eight heterotopic heart transplants were performed in two groups of mice: syngeneic C3H/HeN to C3H/HeN mice and allogeneic BALB/C to C3H/HeN mice. Transplants were followed out to 2, 6, 10, and 14 days. Six transplants were performed in each group. Serum and splenic samples were used to evaluate the cytokine response by ELISA. Explanted heart tissue was processed for evidence of histologic rejection, and RT-PCR was performed to evaluate the IL-12, IL-18, and IFN-gamma signal qualitatively. Analysis of variance (ANOVA), Fisher's projected least significant difference (PLSD) was used for statistical analysis. Transplant rejection occurred in the allogeneic group histologically by day 6 and clinically by day 10. Serum IFN-gamma levels rose significantly by day 6 in the allogeneic group and then continued to rise in the splenocyte cultures. Serum IL-18 also rose significantly in the allogeneic group at day 6 compared with syngeneic group. RT-PCR revealed that the allogeneic tissue contained an increased signal for IL-12, IL-18, and IFN-gamma beginning at day 6 and peaking at day 10 after transplant. Beginning 6 days after transplantation, IL-12 and IL-18 appear to play a significant role in the induction of IFN-gamma in allogeneic heart transplants.

Coronary artery bypass grafting with cardiopulmonary bypass versus off-pump cardiopulmonary bypass grafting: does eliminating the pump reduce morbidity and cost?

BACKGROUND: Cardiopulmonary bypass (CPB) may contribute to the complications and cost of coronary artery bypass grafting (CABG). Off-pump CABG (OPCAB) allows coronary revascularization without CPB. We hypothesized that OPCAB provides satisfactory graft patency while reducing complications and cost compared with CABG with CPB. METHODS: We prospectively followed 80 patients undergoing CABG: 40 patients undergoing OPCAB and 40 patients undergoing CABG with CPB. OPCAB patients underwent angiography within 48 hours of surgery to determine early graft patency. Incidence of complications, length of stay, and costs were recorded for each patient. The influence of the number of vessels bypassed was analyzed. RESULTS: OPCAB patients (n = 40) underwent grafting of 2.7 +/- 0.7 vessels per patient compared with 3.6 +/- 0.8 vessels per patient in the CABG with CPB group (n = 40) (p < 0.0001). Angiography demonstrated 105 of 108 (97%) of grafts were patent in the OPCAB group. Incidence of complications, length of stay, and costs did not differ between the OPCAB and CABG with CPB groups. Number of vessels grafted showed a positive correlation to total costs in both groups. CONCLUSIONS: While OPCAB provided satisfactory early graft patency, there was no significant difference between OPCAB and CABG with CPB with regard to cost, length of stay, or incidence of complications. In this study, eliminating CPB did not reduce morbidity or cost after CABG.

Improved biochemical preservation of lung slices during cold storage.

BACKGROUND: Development of lung preservation solutions typically requires whole-organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that lung slices could be used to assess preservation of biochemical function during cold storage. MATERIALS AND METHODS: Whole rat lungs were precision cut into slices with a thickness of 500 microm and preserved at 4 degrees C in the following solutions: University of Wisconsin (UW), Euro-Collins (EC), low-potassium-dextran (LPD), Kyoto (K), normal saline (NS), or a novel lung preservation solution (NPS) developed using this model. Lung biochemical function was assessed by ATP content (etamol ATP/mg wet wt) and capacity for protein synthesis (cpm/mg protein) immediately following slicing (0 h) and at 6, 12, 18, and 24 h of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as means +/- SD. RESULTS: ATP content was significantly higher in the lung slices stored in NPS compared with all other solutions at each time point (P < 0.0001). Protein synthesis was significantly higher in the lung slices stored in NPS compared with all other solutions at 6, 12, and 18 h of preservation (P < 0.05). CONCLUSIONS: This lung slice model allows the rapid and efficient screening of lung preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel solution that improves the biochemical preservation of lung slices during cold storage.

Aprotinin preserves myocardial biochemical function during cold storage through suppression of tumor necrosis factor.

OBJECTIVE: Inflammatory cytokines, particularly tumor necrosis factor, contribute to myocardial dysfunction after ischemia-reperfusion injury. Aprotinin may improve outcomes in cardiac surgery through suppression of inflammatory mediators. We hypothesized that aprotinin may exert its beneficial effects through suppression of tumor necrosis factor alpha. METHODS: Adult rat hearts were precision cut into slices with a thickness of 200 microm and stored in crystalloid cardioplegic solution alone or with one of the following additions: aprotinin or tumor necrosis factor alpha, aprotinin plus tumor necrosis factor alpha, a monoclonal antibody to tumor necrosis factor alpha, or a polyclonal antibody to the tumor necrosis factor alpha receptor. Myocardial biochemical function was assessed by adenosine triphosphate content and capacity for protein synthesis immediately after slicing (0 hours) and after 2, 4, and 6 hours of storage at 4 degrees C. The content of tumor necrosis factor alpha was measured by an enzyme-linked immunosorbent assay. Six slices were assayed at each time point for each solution. The data were analyzed by analysis of variance and are expressed as the mean +/- standard deviation. RESULTS: When stored in cardioplegic solution containing aprotinin, the heart slices demonstrated (1) an increase in adenosine triphosphate content and protein synthesis (P <.0001), (2) a decrease in intramyocardial generation of tumor necrosis factor alpha (P

Functional outcome and survival after pharyngolaryngoesophagectomy for cancer.

BACKGROUND: Surgical resection of the larynx, hypopharynx and cervical esophagus, or pharyngolaryngoesophagectomy (PLE), with pharyngogastric anastomosis (PGA) offers a means of controlling local and regional carcinoma of the upper aerodigestive tract (UADT). We reviewed our experience with PLE for carcinoma of the UADT to evaluate functional outcome and survival. METHODS: Patients undergoing PLE from 1986 through 1999 were reviewed. Survivors completed questionnaires which graded their level of function and voice rehabilitation. Gastric emptying studies were performed with rates compared with normal controls. Survival curves were generated using the Kaplan-Meier method. RESULTS: Thirty-one patients underwent PLE during the study period. Twenty-nine patients had squamous cell carcinoma. Operative mortality was 0%. Thirty-day mortality was 9.6%. There were 2 anastomotic leaks (6.4%). All survivors reported normal ability to complete activities of daily living. Voice rehabilitation was acceptable in 7 of 10 survivors. Positive surgical margins resulted in decreased survival (P = 0.03). No other patient demographic or management variable altered survival. One-year, 5-year, and 10-year survival rates were 67%, 40%, and 18%, respectively. CONCLUSION: PLE with PGA for carcinoma of the UADT may be performed with low morbidity and mortality. Functional patient outcomes including gastric emptying, activities of daily living, and voice rehabilitation are acceptable.

Improved biochemical preservation of heart slices during cold storage.

BACKGROUND: Development of myocardial preservation solutions requires the use of whole organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that myocardial slices could be used to assess preservation of biochemical function during cold storage. MATERIALS AND METHODS: Whole rat hearts were precision cut into slices with a thickness of 200 microm and preserved at 4 degrees C in one of the following solutions: Columbia University (CU), University of Wisconsin (UW), D5 0.2% normal saline with 20 meq/l KCL (QNS), normal saline (NS), or a novel cardiac preservation solution (NPS) developed using this model. Myocardial biochemical function was assessed by ATP content (etamoles ATP/mg wet weight) and capacity for protein synthesis (counts per minute (cpm)/mg protein) immediately following slicing (0 hours), and at 6, 12, 18, and 24 hours of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as the mean +/- standard deviation. RESULTS: ATP content was higher in the heart slices stored in the NPS compared to all other solutions at 6, 12, 18 and 24 hours of cold storage (p < 0.05). Capacity for protein synthesis was higher in the heart slices stored in the NPS compared to all other solutions at 6, 12, and 18 hours of cold storage (p < 0.05). CONCLUSIONS This myocardial slice model allows the rapid and efficient screening of cardiac preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel preservation solution which improves the biochemical function of myocardial slices during cold storage.

Avoidance of cellular blood product transfusions in LVAD recipients does not prevent HLA allosensitization.

BACKGROUND: Transfusion of cellular blood products during left ventricular assist device (LVAD) implantation has been associated with HLA allosensitization, resulting in the need for a negative prospective cross-match and prolonged transplant waiting times. In order to prevent this risk, we developed a protocol to avoid transfusion of cellular blood products. METHODS: The protocol included preoperative patient stabilization, perioperative recombinant erythropoietin and blood conservation strategies, and postoperative monitoring of mixed venous oxygen saturation (SVO2) to assure adequate peripheral oxygen delivery. Panel reactive antibody (PRA) was measured in all patients pre and post LVAD placement to assess HLA sensitization. RESULTS: Seven consecutive patients underwent LVAD implantation without transfusion of blood or platelets, one of whom expired perioperatively. Mean hematocrit was 35.2% preoperatively, and 21.8% postoperatively, reaching a nadir of 20.2%. Postoperative SVO2 was >60% in all patients. In the six survivors, mean hematocrit reach 24.3%, 27.3%, and 33.0% by postoperative day seven, fourteen, and thirty, respectively. PRA in three patients was 0% preoperatively and remained 0% until transplantation after 33, 34, and 50 days of support. In two patients, preoperative PRA was 7% and 17%, dropped to 3% and 0% after thirty days, then progressively rose to 96% and 100% after 60 and 90 days, respectively. In one other patient, preoperative PRA was 0%, remained at 0% after thirty days, then rose to 96% by 60 days. CONCLUSIONS: Avoiding transfusion of cellular blood products in LVAD recipients is safe and well tolerated, but does not universally protect from HLA allosensitization. Other factors may also produce sensitization, such as immunogenic components of the LVAD, soluble antigen in fresh frozen plasma, or latent sensitization which is not initially evident in critically ill and possibly anergic patients.