Optimization of electroporation conditions for Mycobacterium avium.

Successful transformation and subsequent genetic manipulation of Mycobacterium avium requires suitable vectors, efficient transformation systems, and reliable selectable markers. A systematic analysis of the parameters involved in the transformation of M. avium was performed to optimize DNA transfer. Factors examined included the composition of the growth medium, growth medium additives, variations in washing of the bacteria prior to electroporation, and conditions of electroporation. Of the parameters assayed, the frequency of transformation (defined as the number of transformants per 10(6) transformed bacteria) showed the greatest increase with the addition of 1.5% glycine to the M. avium culture medium and the use of higher concentrations of plasmid DNA. The addition of 0.5 M sucrose to the growth medium and wash solution yielded a modest increase in transformation frequency, but more importantly afforded greater consistency of results between different batches of cells with no decrease in transformation yields following freezing and thawing. We also confirmed that gfp could be used as a selective marker for M. avium, even as a single copy integrant, and allowed for rapid discrimination between false and true transformants. Using this protocol, we were able to transform nine of 11 clinical strains of M. avium.

Emergency pneumonectomy for penetrating and blunt trauma.

Emergency pneumonectomy for penetrating and blunt trauma has an attendant high mortality. Patients with major lung injuries presenting with prolonged shock followed by control of bleeding, resuscitation with or without aortic cross-clamping and pneumonectomy have had uniformly unsatisfactory results. From 1972 to 1982, eight patients at the University of Louisville Hospital underwent emergency pneumonectomy. All patients underwent expeditious evaluation, resuscitation, and thoracotomy with pneumonectomy. Three patients died of exsanguination (2 patients had major associated intra-abdominal injuries). Three other patients died due to pulmonary edema and right ventricular failure 2 to 3 hours after hemorrhage had been controlled and intravascular volume restored. Aortic cross-clamping was employed in four patients due to persistent hypovolemia with 100 per cent mortality. Of the two surviving patients, one presented with stable blood pressure and had pneumonectomy for tracheobronchial disruption, while the other had pneumonectomy for tangential laceration of the lung at the hilum. Pulmonary edema and right ventricular failure were responsible for mortality following emergency pneumonectomy and control of hemorrhage and restoration of blood volume. The addition of aortic cross-clamping did not seem to alter survival and may, indeed, hinder therapy due to increased vascular afterload and increased heart failure and pulmonary edema.

Blood flow distribution in infant pigs subjected to surface cooling, deep hypothermia, and circulatory arrest. Deleterious effects in pigs with left-to-right shunts.

Surface cooling, deep hypothermia and circulatory arrest have been used effectively for correction of congenital heart defects in infancy. Which patients are best suited for this technique has not been addressed. The addition of surface cooling to deep hypothermia and circulatory arrest provides homogeneous cooling and avoids swelling due to reperfusion injury after circulatory arrest. However, surface cooling in patients with large left-to-right shunts causes increased peripheral resistance and increased shunting which can result in decreased perfusion of vital organs. The purpose of this study is to measure the effect of a large left-to-right shunt on total organ blood flow distribution in infant piglets during surface cooling, deep hypothermia, and circulatory arrest. Eleven 2-week-old piglets had surface cooling, deep hypothermia, and circulatory arrest for 45 minutes, followed by rewarming and weaning from cardiopulmonary bypass. Microspheres (15 mu) were injected before surface cooling, at 28 degrees C, at 15 degrees C, and after weaning from cardiopulmonary bypass. Group I (five piglets) was the control. Group II (six piglets) had a large (6 mm) left-to-right aortopulmonary shunt established before microsphere injection. Cardiac outputs in both Groups I and II decreased with surface cooling. The distribution of cardiac output in Group I did not change with surface cooling; however, Group II pigs showed marked change in distribution of cardiac output, resulting in decreased renal, visceral, and pulmonary flow (p less than 0.05). Amylase determinations before and after surface cooling, deep hypothermia, and circulatory arrest were unchanged in Group I but elevated in Group II (p less than 0.05). These observations suggest altered cellular metabolism in visceral organs during the period of surface cooling which may be compounded by circulatory arrest and rewarming.

Management of tracheobronchial disruption resulting from blunt trauma.

Delayed diagnosis of tracheobronchial disruption resulting from blunt trauma continues to cause major morbidity and death. At the University of Louisville from 1968 to 1982, 13 patients had tracheobronchial disruption resulting from blunt trauma. All injuries were caused by motor vehicle accidents. Disruptions were located in the trachea in six patients and in the right bronchus in seven patients. Physical findings included: subcutaneous emphysema (11 patients), respiratory distress (10 patients), hemoptysis (six patients), and flail chest (four patients). Four patients (30%) died, three from multiple major associated injuries and the other before therapy could be instituted. Among the nine survivors, six had immediate diagnosis and prompt surgical treatment, which consisted of suture repair in five and pneumonectomy in the other patient. Two patients had delay in diagnosis, and repair was attempted at 4 and 30 days, respectively; bronchial stricture resulted in one and pneumonectomy, empyema, and bronchopleural fistula in the other. Another patient with a bronchial mucosal tear was treated nonoperatively without complication. Tracheobronchial disruption should always be considered with massive blunt chest trauma. Repeated bronchoscopy is indicated for unexplained pleural air leaks, lobar atelectasis, or persistent pneumothorax. Prompt diagnosis and expeditious surgical therapy result in fewer complications and increased survival.

Creation of left-to-right shunts in the newborn pig: a new model.

The closer to birth that congenital heart defects are simulated, the more valid the model. Unlike obstructive lesions such as pulmonic and aortic stenosis, which can be constructed in utero or in infancy, left-to-right shunts are more difficult to create due to the extreme friability of fetal and neonatal tissue. A method for creating left-to-right shunts in infant pigs using a pleural-sparing transverse sternotomy and direct side-to-side anastomosis between the redundant pulmonary artery and the ascending aorta has been introduced. The pigs developed palpable thrills, systolic and diastolic flow murmurs, and congestive heart failure. The average pulmonary-to-systemic flow ratio by the microsphere technique was 2.1:1 compared to 2.1:1 by oximetry. Left-to-right shunts can be constructed in infant pigs to simulate congenital heart defects.

Management of patent ductus arteriosus in the premature infant: indomethacin versus ligation.

A previous report from our institution analyzed the results of pharmacological (indomethacin) closure of patent ductus arteriosus (PDA) in 82 neonates. Closure was achieved in 54 patients. However, gastrointestinal complications occurred in 21, necrotizing enterocolitis in 13, and focal perforation in 8. Overall mortality in the indomethacin group was 40%. This paper compares the results of that pharmacological experience with our subsequent surgical experience with 86 low-birth-weight neonates for whom gestational age, size, illness, and mode of diagnosis were comparable. Mean weight at operation for this study was 1.1 kg; mean gestational age was 29.1 weeks. All infants required endotracheal-assisted ventilation for severe radiographic and clinical hyaline membrane disease. Range-gated Doppler study, retrograde flush aortography, and echocardiographic measurement of the ratio between the left atrium and the aortic root were used to confirm the diagnosis of PDA. Ligation was done in the neonatal intensive care unit using local anesthesia supplemented with morphine. Ventilation was controlled by an inhalation therapist; drug and blood administration were controlled by the infant's nurse. Surgical ligation was employed in all infants except for 7 in whom hemoclip ductal closure was chosen because of extreme instability, coagulopathy, or ductal perforation. There were no operative deaths. Surgical morbidity included ductal perforation (2 patients), wound infection (1), and phrenic nerve injury (1). Necrotizing enterocolitis occurred in 9 patients. The overall mortality was 17%. Patients with preoperative pneumo-thorax had a 32% overall mortality.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstruction of complex thoracic defects with myocutaneous and muscle flaps. Applications of new flap refinements.

This report describes reconstructions of complex thoracic defects with myocutaneous and muscle flaps that were modified by several recent refinements of flap design. These refinements comprise a second generation of myocutaneous and muscle flaps, which have substantially increased versatility and extended applications, as compared with the originally described flaps. These refinements include the following: (1) segmentally split latissimus dorsi and pectoralis major flaps, which transfer only one muscle segment as the flap and leave other segments of the same muscle in situ to preserve motor function; (2) pectoralis major fasciocutaneous flaps, which are extended by abdominal skin and fascia to provide longer, larger flaps; (3) reversed pectoralis major and latissimus dorsi flaps, which are supplied by secondary, distal vascular pedicles that permit flap use when the primary vascular supply is interrupted; and (4) island vascular pedicle muscle flaps, which allow intercostal passage for reconstruction of intrathoracic defects and cavities. The anatomic bases for these flap refinements are described, and the advantages provided are discussed.

Granular cell myoblastoma of the esophagus.

Twenty-seven cases of granular cell myoblastoma of the esophagus have been reported. We describe two additional cases. The clinical aspects are analyzed briefly. Although the malignancy potential is low, invasion does occur, and these lesions should be removed.