Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment.

Maggot debridement therapy (MDT) is a method for the treatment of intractable, infected and necrotic wounds. In MDT, sterile larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) are applied to infected wounds, where they exert antibacterial effects. Once the larvae are placed in the wound, they are no longer germ-free. This study analysed the influence of infected environments on larval antibacterial activities. Sterile larvae were mixed in a test tube containing a bacterial suspension of Staphylococcus aureus or Pseudomonas aeruginosa, transferred to liver puree agar, and incubated at 25 °C for set periods. To collect the larval extracts, the incubated larvae were transferred to a test tube containing phosphate buffered saline (PBS), cut into multiple pieces with scissors, and centrifuged. The supernatant was used to test antibacterial activities. The results showed that infected larvae had better antibacterial capacities than sterile larvae. Antibacterial activities were induced by pretreatment with a single bacterial species, S. aureus or P. aeruginosa, within 24 h and 12 h, respectively, and disappeared after 36 h. The activities were effective against S. aureus, but not against P. aeruginosa. This natural infection model is very similar to the clinical wound context in MDT and will be a powerful tool with which to study the antibacterial activities of L. sericata larvae in MDT.

[Surgical treatment of transposition of great arteries: the coronary buttons transfer using medially based trapdoor flaps].

The arterial switch operation (ASO) has become the primary surgical approach used for correction of transposition of the great arteries. All the prerequisites for a successful ASO were recognized in time and dealt with, which allowed general acceptation of the technique. We report on our technique for the procedure and the result to date. From January 1991 to January 2008, a total of 100 patients underwent ASO at our unit using medially-based trapdoor flap method. The neo-pulmonary artery (PA) was reconstructed using a single rectangular pericardial patch. The initial patient having intramural coronary artery died due to ischemic event after Aubert procedure. Three patients had re-right ventricular out flow tract repair (RVOTR) in a long-term follow-up period. There was no significant aortic insufficiency, no ischemic event and no lethal arrhythmia.

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.

Growth enhancement of normal human keratinocytes by the antisense oligonucleotide of retinoblastoma susceptibility gene.

We have found that the growth of normal human keratinocytes, grown in serum-free medium, was significantly stimulated by the antisense oligonucleotide of retinoblastoma susceptibility gene (Rb). Normal human keratinocytes were exposed to phosphorothionate oligonucleotides which were complementary to translation initiation codon of Rb gene. The growth of keratinocytes was enhanced by the antisense, but not the sense, oligonucleotide of Rb gene in a dose-dependent manner from 1 to 10 microM. The Rb antisense oligonucleotide, however, did not result in any appreciable change in transcription of the gene when examined by reverse-transcription polymerase chain reaction (RT-PCR) analysis or in the protein expression and the phosphorylation pattern when examined by immunoprecipitation and Western blotting.

PKCeta associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes.

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCeta is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCeta that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCeta and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCeta with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCeta with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCeta associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated. Oncogene (2000) 19, 6334 - 6341.

Myocardial adenine nucleotides, glycogen, and Na, K-ATPase in patients with idiopathic dilated cardiomyopathy requiring mechanical circulatory support.

Acute decompensation leading to progressive pump failure is a main cause of death in patients with congestive heart failure. To find possible metabolic defects associated with the onset of this fatal occurrence, we measured myocardial adenine nucleotides, glycogen, and Na,K-ATPase in patients with end-stage idiopathic dilated cardiomyopathy. The biopsy specimens were obtained from the right ventricle of beating hearts during implantation of a biventricular assistance device in 23 patients (group I) suffering from irreversible cardiogenic shock and during heart transplantation in 20 patients (group II) in compensated heart failure. Left ventricular ejection fraction (LVEF) was determined preoperatively by echocardiography. Left ventricular function in group I was more severely impaired than in group II (LVEF 16.8%+/-4.6% vs 22.1%+/-5.1 %; p <0.01). Myocardial adenosine triphosphate (ATP) in group I was significantly reduced in comparison with group II (119.4+/-10.2 vs 27.7+/-7.4 nmol/mg noncollagen protein; p <0.01). There was no difference in glycogen levels. Na,K-ATPase concentration in group I (n = 8) was lower than that of group II (n = 20) (425+/-80 vs 498+/-75 pmol/g wet weight; p <0.05). Linear regression analyses showed a significant correlation between adenosine triphosphate (ATP) and LVEF (r = 0.41, p <0.01) and between Na,K-ATPase and LVEF (r = 0.55, p <0.01). These results indicate that loss of myocardial ATP and Na,K-ATPase could partially contribute to the development of spontaneous deterioration of the chronically overloaded heart.

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes.

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

The modified Norwood procedure for hypoplastic left heart syndrome: early to intermediate results of 120 patients with particular reference to aortic arch repair.

BACKGROUND: Classic first-stage Norwood repair of hypoplastic left heart syndrome uses a homograft patch enlargement to obtain an unobstructed aorta and coronary arteries. Because of possible disadvantages of the homograft, such as lack of growth, degeneration and calcification, and availability, we have tried to repair the aorta without patch supplementation. METHODS: Between February 1993 and September 1997, 120 patients, aged birth to 47 days (median 4 days) and weighing 1.7 to 4.4 kg (median 3.1 kg), underwent first-stage palliation for hypoplastic left heart syndrome. The diameter of the ascending aorta ranged from 1.5 to 8.0 mm (median 3.0 mm). Eight patients had an aberrant right subclavian artery arising from the descending thoracic aorta. In 95 patients (group I), all duct tissue was excised and the descending aorta was anastomosed to the aortic arch, which had been opened back into the ascending aorta. Then to this confluence was anastomosed the proximal main pulmonary artery. In the remaining 25 patients (group II), continuity of the aortic arch was maintained and the repair was performed with a Damus-Kaye-Stansel anastomosis. The size of the systemic-to-pulmonary shunt was 3 mm in 48 patients, 3.5 mm in 70, and 4.0 mm in 2. RESULTS: Circulatory arrest time ranged from 19 to 105 minutes (median 54 minutes). A homograft patch was necessary for the arch reconstruction in 18 patients (15%); 9 group I patients (10%) and 9 group II (36%) (P =.001). There were 82 hospital survivors (68%); 69 group I patients (73%) and 13 group II (52%) (P =.04), 71 patients without a patch (70%) and 11 with a patch (61%) (P >.2). By multiple logistic regression, the aberrant right subclavian artery was a significant risk factor for hospital death (P =.008). There were 6 late deaths. Sixteen of 71 patients (23%) who underwent second-stage palliation had a neoaortic arch obstruction develop, with a peak gradient greater than 10 mm Hg; 14 group I patients (23%) and 2 group II (22%) ( P >.2), 15 without a patch (23%) and 1 with a patch (17%) (P >.2). Overall survivals were 57% at 1 year and 55% at 2 years. CONCLUSION: The modified Norwood procedure for first-stage palliation of hypoplastic left heart syndrome is possible in the majority of patients without the use of exogenous materials and does not result in an increased incidence of neoaortic arch obstruction. Repair of the aorta without patch supplementation may improve the potential for long-term growth of the new aorta.

Staged biventricular repair of pulmonary atresia or stenosis with intact ventricular septum.

BACKGROUND: Since 1991 we have performed a multistage palliative approach to biventricular repair of pulmonary atresia or critical pulmonary stenosis with intact ventricular septum in infants with a detectable right ventricular infundibulum. METHODS: A total of 25 patients (19 pulmonary atresia and 6 critical pulmonary stenosis) underwent initial palliation consisting of a transarterial pulmonary valvotomy and a polytetrafluoroethylene shunt between the left subclavian artery and pulmonary trunk. Among the 23 survivors, 15 underwent balloon valvotomy. Six of these patients later required additional palliative surgery that consisted of repeat pulmonary valvotomy, adjustment of an atrial communication, and resection of the hypertrophied muscles in the right ventricle. RESULTS: Of the 25 patients, 23 (92%) survived. In all, 20 patients underwent definitive operations: 18 (90%) biventricular repair (12 pulmonary atresia, and 6 critical pulmonary stenosis), one bidirectional Glenn, and one Fontan procedure. The actuarial probability of achieving a biventricular repair at 36 months of age was 69%. In 18 patients right ventricular end-diastolic volume significantly increased but tricuspid valve diameter did not change. CONCLUSIONS: The multistage palliation procedure to promote right ventricular growth makes a definitive biventricular repair of pulmonary atresia or critical pulmonary stenosis with intact ventricular septum possible in the majority of infants with a patent infundibulum.

Correction of complete atrioventricular septal defects with the double-patch technique and cleft closure.

BACKGROUND: Controversy continues to surround determining which is the most beneficial method of complete atrioventricular septal defect repair, eg, one- versus two-patch repair, closure of mitral cleft, and the necessity of annuloplasty. METHODS: Between January 1988 and November 1995, 120 patients with complete atrioventricular septal defect underwent total correction at the German Heart Institute Berlin. Sixty-nine of the patients were infants and 51 were children or adolescents. Eleven patients had previously undergone pulmonary artery banding. One hundred three patients had Down's syndrome. In all 120 patients complete atrioventricular septal defect repair was performed using the two-patch technique. The mitral cleft was closed with interrupted sutures in 119 cases. RESULTS: Thirty-four patients required aggressive treatment of postoperative pulmonary hypertensive crises (including nitric oxide inhalation). There were 12 hospital deaths (10%). Mortality was highest in patients with persistently high postoperative pulmonary arterial pressure (pulmonary artery pressure/systemic artery pressure > 0.6) (7 of 17 patients died; 41%). Associated atrioventricular valve anomalies, especially dysplastic valve tissue and severe preoperative cardiopulmonary instability necessitating catecholamine support and artificial ventilation, represented other risk factors. There were six late deaths (5%); cumulative mortality was 15%. Four patients suffered a complete heart block and sick sinus node syndrome necessitating pacemaker implantation 1 to 6 months after operation. During the follow-up period (3 to 80 months after operation), 7 patients (6.8% of survivors) were successfully reoperated on after significant mitral valve incompetence due to an open "cleft" (suture failure) developed. CONCLUSIONS: Correcting complete atrioventricular septal defect using the two-patch technique, routine cleft closure, and atrial septal incision led to a low incidence of residual mitral valve incompetence. Mortality was primarily influenced by severe cardiopulmonary instability and additional atrioventricular valve anomalies preoperatively and the persistence of high pulmonary arterial hypertension postoperatively.

Myocardial recovery through ECMO after repair of total anomalous pulmonary venous connection: the importance of left heart unloading.

A 7-day-old boy who had been placed on extracorporeal membrane oxygenation on his second day of life developed biventricular failure after undergoing surgical repair of a supracardiac variant of total anomalous pulmonary venous connection. Extracorporeal membrane oxygenation was again necessary for postoperative cardiopulmonary support. However, severe left ventricular failure made it imperative to leave the vertical vein open during support in order to decrease pressure on the left ventricle. The patient was successfully weaned from extracorporeal membrane oxygenation on day 8 after surgery and discharged from the hospital on day 23.

Single-stage repair of aortic coarctation with ventricular septal defect using isolated cerebral and myocardial perfusion.

OBJECTIVE: To avoid hypothermic circulatory arrest, we have repaired aortic coarctation with ventricular septal defect (VSD) in a one-stage procedure using an isolated cerebral and myocardial perfusion technique, and retrospectively compared this novel approach to the conventional two-stage approach. METHODS: Between October 1991 and February 1999, 24 infants, aged 4-137 days (median, 27 days) and weighing 1.7-4.3 kg (median, 3.0 kg), underwent the repair of aortic coarctation with VSD either in one (group I, n=11) or two stages (group II, n=13). In Group I, an arterial cannula for cardiopulmonary bypass was inserted into the ascending aorta in six patients with coarctation only, or into a polytetrafluoroethylene (PTFE) graft which was anastomosed to the innominate artery in the remaining five who had hypoplastic arches. A cross-clamp was placed between the innominate and left carotid arteries. The bypass flow was reduced to 30-50% of full flow at 28 degrees C, thereby maintaining a radial artery pressure of 30-45 mmHg. At this point, the aortic coarctation was repaired by an end-to-end arch anastomosis, while maintaining brain perfusion and with the heart still beating. In five patients with hypoplastic aortic arches, the innominate artery proximal to the graft was then secured down and the arch anastomosis was extended to the distal ascending aorta, while providing isolated cerebral perfusion and cardioplegic arrest. After arch reconstruction was performed, the clamp was moved onto the ascending aorta, and the VSD was closed with systemic perfusion. In contrast, for group II patients, coarctation repairs were performed through a posterolateral approach, and existing VSDs were closed as secondary procedures. RESULTS: The mean isolated cerebral and myocardial perfusion time for group I was 13 min (range, 7-20 min). The myocardial ischemic time did not differ between groups I and II (43+/-4 vs. 42+/-5 min, not significant). There were no hospital mortalities or neurological complications in either group, but one late death in each group. CONCLUSION: Single-stage repair of aortic coarctation with VSD does not increase myocardial ischemic time compared to the traditional two-stage approach. The isolated cerebral and myocardial perfusion technique may offer substantial brain and myocardial protection during aortic arch reconstruction.

Successful extracorporeal circulatory support after aortic reimplantation of anomalous left coronary artery.

The development of severe heart failure is the main cause of postoperative mortality after the surgical treatment of anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA). Two patients with ALCAPA who developed low cardiac output and could not be weaned from cardiopulmonary bypass (CPB) after aortic reimplantation of the anomalous left coronary artery were successfully treated with a centrifugal left ventricular assist device (LVAD) and extracorporeal membrane oxygenation (ECMO). The circulation of a 10-month-old infant with severe left ventricular dysfunction was supported 192 h postoperatively with a LVAD and a 9-year-old boy with severe right ventricular failure received ECMO support for 99 h following surgery. Both patients survived and, 4 and 9 months after surgery, are asymptomatic and have normal ventricular function. If CPB (up to 3 h) is not effective in improving ventricular function after surgery for ALCAPA, ECMO or LVAD must be used since myocardial recovery in these patients can occur only after prolonged extracorporeal circulatory support.

Circulatory support with paracorporeal pneumatic ventricular assist device (VAD) in infants and children.

OBJECTIVE: The feasibility and efficacy of the pneumatic 'Berlin Heart' ventricular assist device (VAD) were evaluated in 14 pediatric patients with profound cardiogenic shock refractory to conventional therapy. METHODS: There were two patient groups. Eleven patients, aged 2 weeks 15 years and weighing 3.2-52 kg received a left ventricular assist device or a biventricular assist device as a bridge to cardiac transplantation (bridge group). Nine of them had liver, kidney, or lung dysfunction before device implantation. Three patients were supported with a biventricular assist device for myocardial recovery (recovery group): a 6-month-old girl for postcardiotomy shock, a 10-month-old girl for allograft failure after cardiac transplantation, and a 4-year-old boy with acute myocarditis. RESULTS: In the bridge group, eight patients were transplanted after a bridge duration of 6-98 days (mean, 32 days) with five long-term survivors. Organ functions were normalized during bridging in all of the transplant recipients. In the recovery group, the first patient was removed from support after 2 days because of irreversible brain damage. The second patient was weaned from biventricular support after 8 days, but suffered from recurrent allograft failure. The third patient received biventricular support for 21 days followed by extracorporeal membrane oxygenation and was subsequently discharged from the hospital. CONCLUSIONS: The 'Berlin Heart' VAD can keep selected infants and children with life-threatening heart failure for weeks or months.

Mechanical circulatory support for post cardiotomy cardiogenic shock in infants.

Eleven infants weighing 2.3 to 7.8 kg underwent mechanical circulatory support for post cardiotomy cardiogenic shock. Initiated pre-operatively in two patients, extracorporeal membrane oxygenation was used in a total of eight patients aged 6 days to 3 months in association with repair of cyanotic congenital heart disease with increased pulmonary blood flow or with a right sided obstructive lesion. Ventricular assist devices were used in three other patients: a centrifugal left ventricular assist device in Patient 1 (10 months, 5.7 kg) after repair of the anomalous left coronary artery, and a pneumatic biventricular assist device (stroke volume 12 ml) in Patient 2 (6 months, 7.0 kg) for cardiac arrest after closure of ventricular septal defect and in Patient 3 (10 months, 7.8 kg) for post transplant graft failure. Duration of extracorporeal membrane oxygenation duration ranged from 26 to 192 hr (mean, 88 hr). Three patients were weaned from extracorporeal membrane oxygenation and two survived. Two others were separated from extracorporeal membrane oxygenation because of bleeding, but both subsequently died. Patient 1 was weaned from the left ventricular assist device after 192 hr and discharged from the hospital. Support was discontinued after 45 hr in Patient 2 who exhibited irreversible brain damage. Patient 3 was weaned from a biventricular assist device after 174 hr, but suffered recurrent graft failure. Our results show that an appropriate circulatory support system should be selected according to the cardiac anatomy in infants.

Determination of the isoelectric point value of 3-mercaptopyruvate sulfurtransferase and its shift by treatment with oxidized glutathione.

The isoelectric point (pI) value of 3-mercaptopyruvate sulfurtransferase (MST) from human erythrocytes was determined to be 6.3 at 10 degrees C by isoelectric focusing in horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). The value was determined by comparison with the electrofocused bands of bovine pancreatic ribonuclease A-glutathione mixed disulfides (RNase-SG), which were composed of 8 species containing 1 (RNase-SG1) through 8 (RNase-SG8) moles of glutathione per mole of ribonuclease A with different pI values ranging from 5.3 (RNase-SG8) to 8.8 (RNase-SG1). The pI value of the same enzyme in a 110,000 X g supernatant of rat liver was 5.9, which was the same as that of rat erythrocyte enzyme. Treatments of rat hemolysate with oxidized glutathione or diamide resulted in a shift of the pI of MST to a lower value, 5.7-5.5. This shift was inhibited when these treatments were performed in the presence of dithiothreitol. These results indicate that the treatment of the enzyme with oxidized glutathione results in the formation of enzyme-glutathione mixed disulfide.

Preparation of a volatile derivative of taurine and application to gas chromatographic determination of urinary taurine.

A new volatile derivative of taurine, N-isobutoxycarbonyltaurine methyl ester (methyl 2-(N-isobutoxycarbonylamino)ethanesulfonate), was prepared by a three-step procedure for the gas chromatographic determination of taurine in urine. First, taurine was converted to its silver salt by reaction with silver oxide; next the silver salt was reacted with isobutyl chloroformate to form the N-isobutoxycarbonyl derivative, and finally the derivative was reacted with methyl iodide to form N-isobutoxycarbonyltaurine methyl ester. The volatile derivative was analyzed by gas chromatography using a column of 3% OV-101 on Chromosorb W. When methyl 3-(N-isobutoxycarbonylamino) propanesulfonate was used as an internal standard, the calibration curve was linear between 0.5 and 5.0 mumol of taurine/ml and showed a good reproducibility. This method was applied to the determination of taurine in human urine. Recovery was 98.6 +/- 5.2%, when 1.25 to 5.0 mumol/ml of taurine was added to human urine.

Successful use of the centrifugal ventricular assist device for postcardiotomy cardiogenic shock.

A centrifugal pump was successfully used as a left ventricular assist device (LVAD) in a 54-year-old female who developed cardiogenic shock following open heart surgery. Cardiac index prior to the LVAD support was 1.4 l/min/m2 and increased to 3.0 l/min/m2 at removal of the device, which assisted for 88h. She resumed her daily activity 10 months after the operation and is in New York Heart Association functional class I.

Assessment of myocardial function during mechanical left ventricular support using serial echocardiography: a case report.

A 40-year-old man with valvular heart disease was successfully treated using a left ventricular assist device (LVAD) after open heart surgery. Echocardiography revealed left ventricular ejection fraction (LV-EF) at LVAD on/off: 23.4%/14.6% on the 4th, 23.8%/23.8% on the 5th, and 23.8%/26.8% on the 6th postoperative day (POD), respectively. The patient was weaned from LVAD on the 8th POD and discharged from the hospital on the 58th POD. The LV-EF improved to 54% 6 months after surgery and increased from 57% to 64% in response to exercise stress testing 1 year after surgery.

Mechanical circulatory support with a centrifugal pump after open heart surgery.

Since December 1988, a centrifugal ventricular assist device (VAD) was used to support the circulation in 5 patients who could not be weaned from cardiopulmonary bypass (CPB) or developed cardiogenic shock after removal from CPB. Three patients required a left VAD, one needed a right VAD. One patient had biventricular support using a centrifugal left VAD and a diaphragm type right VAD. The duration of the centrifugal VAD support ranged from 6 to 136 (mean 72)h. All patients were weaned from the VAD, but only 2 patients were discharged from the hospital. Two patients died of multiple organ failure, and one died of cardiogenic shock caused by intractable arrhythmia. Infection occurred in all non-survivors, and 2 of them developed renal failure. We conclude that the centrifugal VAD is effective to recover a failing ventricle. The factors related to the unsuccessful recovery were delayed start of the VAD support and major complications such as infection as infection and renal failure.