Plateaus in amyotrophic lateral sclerosis progression: results from a population-based cohort.

BACKGROUND AND PURPOSE: The aim was to assess the frequency of plateaus in amyotrophic lateral sclerosis (ALS) progression using a large population-based cohort. METHODS: Data from the Piemonte and Valle d'Aosta ALS register were used. Patients who were diagnosed between 2007 and 2014 were considered. The follow-up period was extended until 31 December 2018. Visits after tracheostomy were excluded. A plateau was defined as a stable Amyotrophic Lateral Sclerosis Functional Rating Scale revised (ALSFRSr) score lasting at least 6, 12 or 18 months. RESULTS: Out of 1214 patients, 200 (16.5%), 93 (7.7%) and 52 (4.3%) showed at least one plateau lasting a minimum of 6, 12 and 18 months, respectively. Plateaus occurred mostly at high ALSFRSr scores and were more frequent during the initial phases of the disease course. Spinal onset [odds ratio (OR) 1.83, 95% confidence interval (CI) 1.16-2.95, P value 0.01) and predominant upper motor neuron phenotype (OR 2.18, 95% CI 1.36-3.48, P value 0.001) conferred a higher risk for the subsequent appearance of plateaus; conversely, older age at diagnosis (OR 0.25, 95% CI 0.11-0.54, P value 0.002 for >75 year age class) reduced this risk. CONCLUSIONS: Plateaus in ALS progression lasting at least 6 months appear in about one out of six patients and could last even 12, 18 months or more in a smaller subgroup of patients. Plateau occurrence should not lead the neurologist to automatically reconsider ALS diagnosis and should be considered for future clinical trial design.

The role of pre-morbid diabetes on developing amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The literature on the association between diabetes and amyotrophic lateral sclerosis (ALS) consists of a limited number of studies. This cohort study was developed in order to assess the role of diabetes on the risk of developing ALS. METHODS: The study population was represented by all residents in Turin (Italy) at the beginning of 1996 who participated in the 1991 census, over 14 years of age (n = 727 977) and followed up for ALS occurrence from 1998 to 2014. Presence of diabetes at baseline or during follow-up was ascertained through two Piedmont regional sources: the Diabetes Registry and the Anatomical Therapeutic Chemical Drug Prescription Archive. The risk of ALS was estimated using the Piedmont and Valle d'Aosta ALS Registry (PARALS). The association of diabetes, treated as a time-dependent variable, with ALS onset was estimated through Cox proportional hazard regression models adjusted for age, gender, education and marital status. RESULTS: During follow-up, 397 subjects developed ALS, 24 of whom were already diabetic before ALS onset. Diabetes was associated with a significantly decreased risk of ALS [hazard ratio, 0.30 (95% confidence interval, 0.19-0.45)] without differences in risk by gender, age class or ALS phenotype. CONCLUSION: The results support the protective role of diabetes toward ALS.

Spatial epidemiology of amyotrophic lateral sclerosis in Piedmont and Aosta Valley, Italy: a population-based cluster analysis.

BACKGROUND AND PURPOSE: The analysis of the spatial distribution of cases could give important cues on putative environmental causes of a disease. Our aim was to perform a spatial analysis of an amyotrophic lateral sclerosis (ALS) cohort from the Piedmont and Aosta Valley ALS register (PARALS) over a 20-year period. METHODS: The address at the moment of diagnosis was considered for each ALS case. Municipalities' and census divisions' resident populations during the 1995-2014 period were obtained. A cluster analysis was performed adopting both Moran's index and the Kulldorff spatial scan statistic. RESULTS: A total of 2702 ALS patients were identified. An address was retrieved for 2671 (99%) patients. Moran's index was -0.01 (P value 0.83), thus revealing no clusters. SaTScan identified no statistically significant clusters. When census divisions were considered, Moran's index was 0.13 (P value 0.45); SaTScan revealed one statistically significant small cluster in the province of Alessandria. Here, 0.0099 cases were expected and three cases were observed (relative risk 304.60; 95% confidence interval 109.83-845.88, P value 0.03). DISCUSSION: Our study showed a substantial homogeneous distribution of ALS cases in Piedmont and Aosta Valley. The population-based setting and the adoption of proper statistical analyses strengthen the validity of our results. Such a finding further suggests the involvement of multiple environmental and genetic factors in ALS pathogenesis.

Peripheral Blood Gene Expression Changes Associated With Primary Graft Dysfunction After Lung Transplantation.

Recipient responses to primary graft dysfunction (PGD) after lung transplantation may have important implications to the fate of the allograft. We therefore evaluated longitudinal differences in peripheral blood gene expression in subjects with PGD. RNA expression was measured throughout the first transplant year in 106 subjects enrolled in the Clinical Trials in Organ Transplantation-03 study using a panel of 100 hypothesis-driven genes. PGD was defined as grade 3 in the first 72 posttransplant hours. Eighteen genes were differentially expressed over the first year based on PGD development, with significant representation from innate and adaptive immunity genes, with most differences identified very early after transplant. Sixteen genes were overexpressed in the blood of patients with PGD compared to those without PGD within 7 days of allograft reperfusion, with most transcripts encoding innate immune/inflammasome-related proteins, including genes previously associated with PGD. Thirteen genes were underexpressed in patients with PGD compared to those without PGD within 7 days of transplant, highlighted by T cell and adaptive immune regulation genes. Differences in gene expression present within 2 h of reperfusion and persist for days after transplant. Future investigation will focus on the long-term implications of these gene expression differences on the outcome of the allograft.

Occupations and amyotrophic lateral sclerosis: are jobs exposed to the general public at higher risk?

Background: Aim of this study was to assess whether previous employment in certain occupations could be a risk factor for Amyotrophic Lateral Sclerosis (ALS) incidence. This topic has been explored by several studies, but no risk factor has been firmly identified. Methods: The study population consisted of all subjects over 30 years old resident in Turin in 1996 who worked or were unemployed at 1991 Italian census (n = 284 406), followed up for ALS occurrence from 1996 to 2014. The risk of ALS was estimated in relation to the occupation held in 1991, using the Italian classification of occupations at the greatest detail. The association between occupations and ALS risk was estimated through Huber-White sandwich multivariate Poisson regression models adjusted for age, gender, education and marital status. Results: During the follow-up, 208 subjects developed ALS. ALS risk was significantly associated with previous employment as bank teller (IRR = 7.33), general practitioner (IRR = 4.61) and sales representative (IRR = 3.06). Categorizing all occupations as exposed or unexposed to direct contact with general public, it was found that previous employment in this group of occupations increased significantly ALS risk (IRR = 1.51), mainly driven by occupations in direct contact with customers (IRR = 1.79). Conclusions: The study results indicate that ALS risk may be increased by previous employment in occupations implying direct contact with the general public, in particular customers. A possible explanation of this finding, partly supported by the literature, is that workers in contact with the public could be more exposed to certain infections, which would increase their ALS risk.

Donor age and early graft failure after lung transplantation: a cohort study.

Lungs from older adult organ donors are often unused because of concerns for increased mortality. We examined associations between donor age and transplant outcomes among 8860 adult lung transplant recipients using Organ Procurement and Transplantation Network and Lung Transplant Outcomes Group data. We used stratified Cox proportional hazard models and generalized linear mixed models to examine associations between donor age and both 1-year graft failure and primary graft dysfunction (PGD). The rate of 1-year graft failure was similar among recipients of lungs from donors age 18-64 years, but severely ill recipients (Lung Allocation Score [LAS] >47.7 or use of mechanical ventilation) of lungs from donors age 56-64 years had increased rates of 1-year graft failure (p-values for interaction = 0.04 and 0.02, respectively). Recipients of lungs from donors <18 and ≥65 years had increased rates of 1-year graft failure (adjusted hazard ratio [HR] 1.23, 95% CI 1.01-1.50 and adjusted HR 2.15, 95% CI 1.47-3.15, respectively). Donor age was not associated with the risk of PGD. In summary, the use of lungs from donors age 56 to 64 years may be safe for adult candidates without a high LAS and the use of lungs from pediatric donors is associated with a small increase in early graft failure.

Pilot study exploring lung allograft surfactant protein A (SP-A) expression in association with lung transplant outcome.

Primary graft failure and chronic lung allograft dysfunction (CLAD) limit lung transplant long-term outcomes. Various lung diseases have been correlated with surfactant protein (SP) expression and polymorphisms. We sought to investigate the role of SP expression in lung allografts prior to implantation, in relation to posttransplant outcomes. The expression of SP-(A, B, C, D) mRNA was assayed in 42 allografts. Posttransplant assessments include pulmonary function tests, bronchoscopy, broncho-alveolar lavage fluid (BALF) and biopsies to determine allograft rejection. BALF was assayed for SP-A, SP-D in addition to cytokines IL-8, IL-12 and IL-2. The diagnosis of CLAD was evaluated 6 months after transplantation. Lung allografts with low SP-A mRNA expression prior to implantation reduced survival (Log-rank p < 0.0001). No association was noted for the other SPs. Allografts with low SP-A mRNA had greater IL-2 (p = 0.03) and IL-12 (p < 0.0001) in the BALF and a greater incidence of rejection episodes (p = 0.003). Levels of SP-A mRNA expression were associated with the SP-A2 polymorphisms (p = 0.015). Specifically, genotype 1A1A(0) was associated with lower SP-A mRNA expression (p < 0.05). Lung allografts with low levels of SP-A mRNA expression are associated with reduced survival. Lung allograft SP-A mRNA expression appears to be associated with SP-A gene polymorphisms.

Donor surfactant protein D (SP-D) polymorphisms are associated with lung transplant outcome.

Chronic lung allograft dysfunction (CLAD) is the major factor limiting long-term success of lung transplantation. Polymorphisms of surfactant protein D (SP-D), an important molecule within lung innate immunity, have been associated with various lung diseases. We investigated the association between donor lung SP-D polymorphisms and posttransplant CLAD and survival in 191 lung transplant recipients consecutively transplanted. Recipients were prospectively followed with routine pulmonary function tests. Donor DNA was assayed by pyrosequencing for SP-D polymorphisms of two single-nucleotide variations altering amino acids in the mature protein N-terminal domain codon 11 (Met(11) Thr), and in codon 160 (Ala(160) Thr) of the C-terminal domain. CLAD was diagnosed in 88/191 patients, and 60/191 patients have died. Recipients of allografts that expressed the homozygous Met(11) Met variant of aa11 had significantly greater freedom from CLAD development and better survival compared to those with the homozygous Thr(11) Th variant of aa11. No significant association was noted for SP-D variants of aa160. Lung allografts with the SP-D polymorphic variant Thr(11) Th of aa11 are associated with development of CLAD and reduced survival. The observed genetic differences of the donor lung, potentially with their effects on innate immunity, may influence the clinical outcomes after lung transplantation.

Prevalence and clinical picture of gastroesophageal prolapse in gastroesophageal reflux disease.

The prevalence of gastroesophageal (GE) mucosal prolapse in patients with gastroesophageal reflux disease (GERD) was investigated as well as the clinical profile and treatment outcome of these patients. Of the patients who were referred to our service between 1980 and 2008, those patients who received a complete diagnostic work-up, and were successively treated and followed up at our center with interviews, radiology studies, endoscopy, and, when indicated, esophageal manometry and pH recording were selected. The prevalence of GE prolapse in GERD patients was 13.5% (70/516) (40 males and 30 females with a median age of 48, interquartile range 38-57). All patients had dysphagia and reflux symptoms, and 98% (69/70) had epigastric or retrosternal pain. Belching decreased the intensity or resolved the pain in 70% (49/70) of the cases, gross esophagitis was documented in 90% (63/70) of the cases, and hiatus hernias were observed in 62% (43/70) of the cases. GE prolapse in GERD patients was accompanied by more severe pain (P < 0.05) usually associated with belching, more severe esophagitis, and dysphagia (P < 0.05). A fundoplication was offered to 100% of the patients and was accepted by 56% (39/70) (median follow up 60 months, interquartile range 54-72), which included two Collis-Nissen techniques for true short esophagus. Patients who did not accept surgery were medically treated (median follow up 60 months, interquartile range 21-72). Persistent pain was reported in 98% (30/31) of medical cases, belching was reported in 45% (14/31), and GERD symptoms and esophagitis were reported in 81% (25/31). After surgery, pain was resolved in 98% (38/39) of the operative cases, and 79% (31/39) of them were free of GERD symptoms and esophagitis. GE prolapse has a relatively low prevalence in GERD patients. It is characterized by epigastric or retrosternal pain, and the need to belch to attenuate or resolve the pain. The pain is allegedly a result of the mechanical consequences of prolapse of the gastric mucosa into the esophagus.

Cross-disciplinarity in the advance of Antarctic ecosystem research.

The biodiversity, ecosystem services and climate variability of the Antarctic continent and the Southern Ocean are major components of the whole Earth system. Antarctic ecosystems are driven more strongly by the physical environment than many other marine and terrestrial ecosystems. As a consequence, to understand ecological functioning, cross-disciplinary studies are especially important in Antarctic research. The conceptual study presented here is based on a workshop initiated by the Research Programme Antarctic Thresholds - Ecosystem Resilience and Adaptation of the Scientific Committee on Antarctic Research, which focussed on challenges in identifying and applying cross-disciplinary approaches in the Antarctic. Novel ideas and first steps in their implementation were clustered into eight themes. These ranged from scale problems, through risk maps, and organism/ecosystem responses to multiple environmental changes and evolutionary processes. Scaling models and data across different spatial and temporal scales were identified as an overarching challenge. Approaches to bridge gaps in Antarctic research programmes included multi-disciplinary monitoring, linking biomolecular findings and simulated physical environments, as well as integrative ecological modelling. The results of advanced cross-disciplinary approaches can contribute significantly to our knowledge of Antarctic and global ecosystem functioning, the consequences of climate change, and to global assessments that ultimately benefit humankind.

Life expectancy in patients with hereditary haemorrhagic telangiectasia.

BACKGROUND: There are few data on life expectancy in patients with hereditary haemorrhagic telangiectasia (HHT), a disorder with life-threatening complications. METHODS: Seventy HHT patients provided data on age and age at death of their HHT-affected parent, which was compared with that of the parent's non-affected partner. RESULTS: At the time of the study, 40 HHT parents (57.1%) vs. 36 (51.4%) non-HHT parents had died (p = 0.404). Median age at death was lower in HHT vs. non-HHT parents (63.2 vs. 70.0 years, respectively). The mortality of HHT parents showed an early peak in the under 50s and a late peak at 60-79 years. HHT was the main risk factor influencing life expectancy after 30 years (p < 0.05). No differences in survival probability were found in HHT patients with respect to sex (p = 0.37), or ENG vs. ALK-1 genotype (p < 0.9). DISCUSSION: Life expectancy appears to be significantly lower in HHT patients than in their partners. Prevention of HHT complications with screening programs could increase life expectancy.

Comparison between subjective and objective assessment of the long-term results after the Heller-Dor operation in patients affected by oesophageal achalasia.

INTRODUCTION: Nowadays the subjective assessment of Health-Related Quality of Life after surgery for achalasia is often associated with the instrumental methods in order to evaluate long-term results of therapy. AIMS: To assess the long-term objective and subjective results of the surgical treatment of achalasia and to study the correlation between clinical-instrumental methods and those based on the patient's self-assessment and on Health-Related Quality of Life questionnaires. METHODS: One hundred and twenty-four patients consecutively submitted to trans-abdominal Heller-Dor operation were periodically followed up with clinical examination, endoscopy, barium swallow and manometry. The Health-Related Quality of Life was assessed using the 36 item short form (SF-36) and the Psychological General Well-Being Index questionnaire. The statistical comparison between the results of the self-assessment questionnaires and the long-term clinical-instrumental result was calculated by means of linear regression analysis. RESULTS: Over the years, 123 patients underwent at least one complete clinical-instrumental check-up and filled the self-assessment questionnaires. Mean follow-up was 105 months (range 12-288) with a median of 82.5 months. The result of the surgery was considered satisfactory in 93.5% of the patients, while the reflux oesophagitis observed in 6.5% of the cases was the main cause of failure. Clinical scores for dysphagia and for gastro-oesophageal reflux symptoms were significantly reduced after surgery. The results of the SF-36 and Psychological General Well-Being Index questionnaires were in our population very high and clinical correlation (p<0.05) emerged in physical function, in role physical, in mental health and in vitality domains of SF-36 questionnaire, and in self-control and general health scales of Psychological General Well-Being Index questionnaire. CONCLUSIONS: Health-Related Quality of Life questionnaires can be considered valid aids in evaluating surgical results, but the clinical-instrumental evaluation remains the cardinal point of every long-term assessment in order to diagnose complications, the disease-related conditions of the patient and to acquire reliable data on which scientific discussion can be based.

Coherent regimes of mutually coupled Chua's circuits.

We study the dynamical regimes that emerge from the strong coupling between two Chua's circuits with parameters mismatch. For the region around the perfect synchronous state we show how to combine parameter diversity and coupling in order to robustly and precisely target a desired regime. This target process allows us to obtain regimes that may lie outside parameter ranges accessible for any isolated circuit. The results are obtained by following a recently developed theoretical technique, the order parameter expansion, and are verified both by numerical simulations and on electronic circuits. The theoretical results indicate that the same predictable change in the collective dynamics can be obtained for large populations of strongly coupled circuits with parameter mismatches.

Review article: indications for anti-reflux surgery in gastro-oesophageal reflux disease.

Gastro-oesophageal reflux disease (GERD) is a complex multifactorial disorder whose treatment is based on knowledge of its pathophysiology, natural history and evolution. Recently the relationship between the severest degrees of cardial incontinence and hiatus hernia has been emphasized, which causes the impairment of the mechanical properties of the gastro-oesophageal barrier and of oesophageal acid clearing. Among different types of hiatus hernia, those characterized by the permanent axial orad migration of the oesophago-gastric (EG) junction (nonreducible hiatus hernia) are correlated with severe GERD. Barium swallow may adequately differentiate hiatal insufficiency, concentric hiatus hernia and short oesophagus which are the steps of migration across or above the diaphragm. When associated with panmural oesophagitis and fibrosis of the oesophageal wall, these conditions may be the cause of recurrence of hiatus hernia and reflux after laparoscopic standard anti-reflux surgical procedures; in the presence of nonreducibility of the EG junction below the diaphragm without tension, dedicated surgical procedures are necessary. It is currently agreed that surgical therapy is indicated for patients affected by severe GERD who are not compliant with long-term medical therapy, require high dosages of drugs and are too young for lifetime medical treatment. While the existence of severe GERD correlated with an irreversible anatomical disorder represents an elective indication for surgery, warrants further investigation. Accurate identification of the functional and anatomical abnormalities underlying GERD is mandatory in order to decide whether medical or surgical therapy should be implemented, and to tailor the surgical technique, laparoscopic or open, to each patient.

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury.

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.

Clinical and surgical relevance of the progressive phases of intrathoracic migration of the gastroesophageal junction in gastroesophageal reflux disease.

OBJECTIVE: The pathophysiologic influence of progressive intrathoracic migration of the gastroesophageal junction axial to the esophagus on gastroesophageal reflux disease was investigated. METHODS: A radiologic-manometric study was performed on hiatal insufficiency, concentric hiatus hernia, and short esophagus, the three radiologic steps of intrathoracic gastroesophageal junction migration, and on healthy volunteers. The distances between inferior and superior margins of the lower esophageal sphincter and the diaphragm were measured. Endoscopic, manometric, and pH-metric evaluations were performed after barium swallow in 38 patients with severe gastroesophageal reflux disease and sliding hiatus hernia with intraabdominally reducible gastroesophageal junction, in 35 patients with hiatal insufficiency, in 40 with concentric hiatus hernia, and in 19 with short esophagus. RESULTS: The distance from the lower esophageal sphincter inferior margin to the diaphragm was different in healthy volunteers (-2.6 +/- 0.9 cm [standard deviation]) versus that in patients with hiatal insufficiency (-1.0 +/- 0.7 cm; p = 0.02), concentric hiatus hernia (-0.8 +/- 1.0 cm; p = 0.02), and short esophagus (4.0 +/- 2.5 cm; p = 0.0002), and in patients with short esophagus versus hiatal insufficiency (p = 0.0002) and concentric hiatus hernia (p = 0.0002). Lower esophageal sphincter tone was reduced between healthy volunteers (19 +/- 9.1 mm Hg [standard deviation]) and patients with sliding hiatus hernia (12 +/- 7.2 mm Hg;p = 0.02), hiatal insufficiency (10 +/- 5.9 mm Hg; p = 0.0001), concentric hiatus hernia (7 +/- 3.1 mm Hg; p = 0.00002), and short esophagus (7 +/- 3.7 mm Hg; p = 0.00003) and between concentric hiatus hernia versus sliding hiatus hernia (p = 0.007). Acid gastroesophageal reflux total time percent was increased between healthy volunteers (2.4% +/- 1.8% [standard deviation]) and patients with sliding hiatus hernia (12.8% +/- 7.8%;p = 0.02), hiatal insufficiency (17.2% +/- 15.8%; p = 0.0001), concentric hiatus hernia (24.0% +/- 19.6%;p = 0.00002), and short esophagus (26.1% +/- 19.6%;p = 0.00002) and between sliding hiatus hernia versus concentric hiatus hernia (p = 0.002) and short esophagus (p = 0.01). CONCLUSIONS: Permanent gastroesophageal junction orad migration axial to the esophagus has greater pathophysiologic relevance on gastroesophageal reflux disease than sliding hiatus hernia with an intraabdominally reducible gastroesophgeal junction. Hiatal insufficiency, concentric hiatus hernia, and short esophagus are markers of progressively increasing irreversible cardial incontinence and therefore indications for surgical therapy.

Patient position for a synchronous cervicothoracoabdominal two-team esophagectomy.

Cervicothoracoabdominal and cervicoabdominal approach are routinely adopted for total or subtotal esophagectomy. We propose a modification of the Nanson's patient position to optimize sequential or simultaneous left cervicotomy, laparotomy, and eventual right thoracotomy with one or two surgical teams. This technique permits better control of the operative field for each phase of the procedure with coordinated operating of two surgical teams on the neck, abdomen, and chest.

Endobronchial transfection of naked TGF-beta1 cDNA attenuates acute lung rejection.

BACKGROUND: We investigated endobronchial transfection of CAT and TGF-beta1 cDNA selectively delivered to the lung graft with or without liposomes. METHODS: Phase I: F344 rats received 130 microg of naked plasmid pCF1-CAT or complexed to liposome GL67 via left main bronchus instillation. Rats were awakened (pCF1-CAT, n = 4; GL67:pCF1-CAT, n = 4) or served as donors in an isogenic transplant (pCF1-CAT, n = 5; GL67:pCF1-CAT, n = 5). ELISA was performed on lungs, hearts, and livers on POD 2. Phase II: BN lungs received TGF-beta1 sense (n = 6); antisense (n = 5); GL67:TGF-beta1 sense (n = 10); or saline solution (n = 10). F344 recipients were sacrificed on POD 5. The arterial pO2 and rejection were assessed. RT-PCR for murine TGF-beta1 was performed. RESULTS: Phase I: CAT expression was 519+/-287 pg and 63+/-68 with pCF1-CAT and 104+/-67 and 37+/-45 with GL67:pCF1-CAT, respectively, in the non-transplant and in the transplant setting. No protein was detected in the hearts, livers, and in the native lung of the recipients. Phase II: RT-PCR confirmed murine TGF-beta1 transfection. pO2 was 362.7+/-110.2 (mean mm Hg +/- SD) for sense TGF-beta1; 146.88+/-85.5 for antisense; 241.5+/-181.5 for GL67-TGF-beta1 sense; and 88.4+/-38.7 for saline. TGF-beta1 sense versus all other groups, p<0.05, GL67-TGF-beta1 sense versus saline, p = 0.01. Rejection was significantly lower for TGF-beta1 sense versus saline, p = 0.04. CONCLUSIONS: Endobronchial administration of naked plasmid achieves selective transfection of lung grafts. Using this strategy, TGF-beta1 reduces early lung allograft rejection.

Efficient naked plasmid cotransfection of lung grafts by extended lung/plasmid exposure time.

BACKGROUND: Multiple gene cotransfection may be an effective strategy to modulate concurrent pathologic events after lung transplantation. We investigated in vivo naked plasmid lung cotransfection during cold preservation and the role of lung parenchyma/naked plasmid exposure time. METHODS: F344 rats underwent left main bronchus instillation of pCF1-CAT (chloramphenicol acetyl transferase) (130 microg) +/- pCF1-beta-Gal (beta-galactosidase) (130 microg) in saline. Part Ia: 4 degrees C preservation versus cotransfection. Lung isografts (4 groups, n = 8) were stored after transfection for 1 (2 groups: one received only pCF1-CAT), 6, and 18 hours. Recipient sacrifice was after 48 hours. Part Ib: 4 degrees C preservation versus transgene expression. Rats were sacrificed 48 hours after transfection in a nontransplant setting (2 groups, n = 8; one received only pCF1-CAT). In a third group (n = 8) lungs were harvested 24 hours after transfection, stored for 18 hours, and recipients were sacrificed after 24 hours. The CAT and beta-Gal enzymatic-linked immunosorbent assays were performed. Part II: Lung/plasmid exposure time. In three groups (n = 6) after pCF1-CAT transfection the left main bronchus was not clamped, clamped for 10 minutes, or clamped for 1 hour. Sacrifice was after 48 hours. RESULTS: Part Ia: Lung CAT protein was (in picograms per 100 microg of total protein): median, 42 (range, 25 to 95) after 1 hour (only CAT); 67 (19 to 296) after 1 hour, 32 (6 to 157) after 6 hours; and 9 (5 to 243) after 18 hours. Lung beta-Gal protein was (in picograms per 100 microg of total protein): median, 20 (range, 5 to 353) after 1 hour; 17 (6 to 157) after 6 hours; 4 (1 to 74) after 18 hours (1 hour versus 18 hours, p = 0.04 for both proteins). CAT and beta-Gal production were significantly correlated (p = 0.0001, r = 0.924). Part Ib: Lung CAT protein was (in picograms per 100 microg of total protein): median, 2 (range, 0.6 to 10) no transplant, only CAT; 7 (0.3 to 13) no transplant; 3 (0.9 to 14) transplant. Part II: Left lung CAT protein was (in picograms per 100 microg of total protein): median, 31 (range, 6 to 83) no clamp; 74 (25 to 430) 10 minutes of clamp; 111 (30 to 263) 1 hour of clamp. Right lung CAT protein was (in picograms per 100 microg of total protein): median, 0.06 (range, 0 to 0.9) no clamp; 1 (0 to 6) 10 minutes of clamp; 1 (0 to 18) 1 hour of clamp. CONCLUSIONS: Efficient lung isograft endobronchial cotransfection results from using naked plasmid. Cold preservation affects transfection efficiency but not transgene expression. Lung parenchyma/naked plasmid exposure time determines transfection efficiency.

Recipient intramuscular gene transfer of active transforming growth factor-beta1 attenuates acute lung rejection.

BACKGROUND: Gene transfer into the donor graft has been demonstrated to be feasible in reducing ischemia-reperfusion injury and rejection in lung transplantation. This study was undertaken to determine whether intramuscular gene transfer into the recipient can also reduce subsequent lung graft rejection. METHODS: Brown Norway rats served as donors and F344 rats as recipients. Recipient animals were injected with 10(10) plaque-forming units of adenovirus encoding active transforming growth factor beta1 (group I, n = 6), beta-galactosidase as adenoviral controls (group II, n = 6), or normal saline without adenovirus (group III, n = 6) into both gluteus muscles 2 days before transplantation. Gene expression was confirmed by enzyme-linked immunosorbent assay. Graft function was assessed on postoperative day 5. RESULTS: Successful gene transfection and expression were confirmed by the presence of active transforming growth factor beta1 protein in muscle and plasma. Oxygenation was significantly improved in group I (group I vs II and III, 353.6 +/- 63.0 mm Hg vs 165.7 +/- 39.9 and 119.1 +/- 41.5 mm Hg; p = 0.02 and 0.004). The muscle transfected with the transforming growth factor beta1 showed granulation tissue with fibroblast accumulation. CONCLUSIONS: Intramuscular adenovirus-mediated gene transfer of active transforming growth factor beta1 into the recipients attenuates acute lung rejection as manifested by significantly improved oxygenation in transplanted lung allografts. This intramuscular transfection approach as a cytokine therapy is feasible in transplantation and may be useful in reducing rejection as well as reperfusion injury.