Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.     

Moderate versus deep hypothermia for the arterial switch operation--experience with 100 consecutive patients.

OBJECTIVES: To evaluate the impact of moderate versus deep perioperative hypothermia on postoperative morbidity in patients receiving the arterial switch operation (ASO). METHODS: One hundred consecutive patients received the ASO from 9/98 to 4/06 using temperature-corrected full-flow moderate (M>24 degrees C, n=51) or deep hypothermic cardiopulmonary bypass (CPB) (D <20 degrees C, n=49). Complex TGA morphology was present in 33 patients (M: 27.4%, D: 38.8%, n.s.). Median age was 9 days (M) versus 10 days (D) and body weight was 3.5+/-0.7 kg (M) versus 3.6+/-0.9 kg (D) (both p=n.s.). Follow-up was 3.7+/-2.1 years. RESULTS: Lowest perioperative rectal temperature was 25.3+/-1.1 degrees C (M) versus 19.0+/-0.8 degrees C (D), p<0.001. Intraoperative blood transfusion (M: 231+/-47 ml, D: 252+/-112 ml, p=0.04) and postoperative lactate level (M: 3.2+/-1.3 mmol/l, D: 3.8+/-2.4 mmol/l, p=0.02) were lower under moderate hypothermia. One patient (D) suffered myocardial ischemia, required ECMO support and died. All other patients were safely weaned from CPB using dopamine (M: 3.0 microg/kg min, D: 3.4 microg/kg min, n.s.) and dobutamine (M: 5.6 microg/kg min, D: 6.7 microg/kg min, p=0.048). Secondary chest closure was performed in 41% (M) versus 59% (D) (p=0.04). Patients were extubated after 89 h (M) versus 126 h (D) (p=0.03). Under moderate hypothermia ICU stay (M: 8.4+/-4.7 days, D: 12.0+/-13.8 days, p=0.03) and hospital stay (M: 12.8+/-6.8 days, D: 20.7+/-15.5 days, p=0.001) were shorter. Five-year freedom from reoperation was 97.0% for simple and 85.2% for complex TGA with RVOT reconstruction in 4/6 patients. CONCLUSIONS: The ASO under full-flow moderate compared to deep hypothermia was advantageous regarding length of procedure and primary chest closure rate. Moderate hypothermia seemed to be beneficial for pulmonary recovery, length of chest tube drainage treatment and inotropic support. No worse early or long-term effects of moderate hypothermia were found.     

Development of bactericidal spinel ferrite nanoparticles with effective biocompatibility for potential wound healing applications

The current study was devised to explore the antibacterial activity and underlying mechanism of spinel ferrite nanoparticles (NPs) along with their biocompatibility and wound healing potentials. In this regard, nickel ferrite and zinc/nickel ferrite NPs were synthesized via a modified co-precipitation method and were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy Energy-dispersive X-ray spectroscopy (EDX). The biocompatibility of the synthesized NPs with human dermal fibroblast (HDF) and red blood cells (RBCs) was assessed. The biocompatible concentrations of the NPs were used to investigate the antimicrobial activity against various pathogenic Gram-negative and Gram-positive bacteria. The mode of bactericidal action was also explored. In vitro scratch assay was performed to evaluate the wound healing potential of NPs. The SEM-EDX analysis showed that the average particles size of nickel ferrite and zinc/nickel ferrite were 49 and 46 nm, respectively, with appropriate elemental composition and homogenous distribution. The XRD pattern showed all the characteristic diffraction peaks of spinel ferrite NPs, which confirmed the synthesis of the pure phase cubic spinel structure. The biocompatible concentration of nickel ferrite and zinc/nickel ferrite NPs was found to be 250 and 125 μg ml⁻¹, respectively. Both the NPs showed inhibition against all the selected strains in the concentration range of 50 to 1000 μg ml⁻¹. Studies on the underlying antimicrobial mechanism revealed damage to the cell membrane, protein leakage, and intracellular reactive oxygen species production. The in vitro scratch assay confirmed the migration and proliferation of fibroblast with artificial wound shrinkage. This study shows that nickel ferrite and zinc/nickel ferrite NPs could be a strong candidate for antibacterial and wound healing nano-drugs.

The current study was devised to explore the antibacterial activity and underlying mechanism of spinel ferrite nanoparticles (NPs) along with their biocompatibility and wound healing potentials.     

Replacing cardiopulmonary bypass with extracorporeal membrane oxygenation in lung transplantation operations.

OBJECTIVE: Cardiopulmonary bypass (CPB) support is required in some lung transplantation (LTX) operations. CPB support and full-dose heparin increases the risks of bleeding and early graft dysfunction. We report our experiences of replacing CPB with heparin-bonded low-dose heparin extracorporeal membrane oxygenation (ECMO) support in LTX surgery. METHODS: From 2003 to 2005 forty-seven patients were transplanted. Thirty-seven LTX patients were retrospectively evaluated for this study (10 patients were excluded due to heart-lung-, lung-kidney transplantation, LTX with bypass grafting, and ASD closure or emergency CPB support). Extracorporeal circulation support was necessary in 40% of the 37 LTX patients due to severe primary or secondary pulmonary hypertension (P or SPHTN), right heart dysfunction, or hemodynamic instability. There were seven LTX procedures with CPB and eight implantations with ECMO support. CPB (high-dose heparin) and ECMO support (ACT 160-220 s) was always set up through femoral veno-arterial canulation. All patients had limited access thoracotomies without transsection of the sternum. Normothermia was maintained in all patients. CPB patients: PPH 15%, COPD 15%, IPF with mean PAP>40 mmHg 70%. ECMO patients: PPH 13%, COPD 13%, IPF with severe PAP pressure elevation 74%. RESULTS: In patients undergoing LTX for PPH, the ECMO support was directly extended into the post-operative period. Packed red blood cell (PRBC) transfusion requirements during the operation and the first 24h were 13.25+/-1.6 PRBC units versus 5.1+/-2.8 PRBC units on CBP (p=0.02). Operative time was longer (p=0.11) in the ECMO LTX (451 min+/-76 vs 346+/-140). The increased 90-day mortality rate of the ECMO patients showed a trend toward significance (p=0.056), which was related to infectious complications (3 vs 1 patient). Severe graft ischemia/reperfusion injury occurred in 9% in the CPB versus 13% in the ECMO group. The 1-year survival was significantly reduced in ECMO patients (p=0.004, log-rank test). CONCLUSIONS: The advantages of femoral canulation rather than conventional central connections in lung transplantation procedures led to an undisturbed operative field. A significantly higher blood product amount was required in ECMO patients, which might lead to increased infection and mortality rates. CPB, obviously, should remain the standard support technique if extracorporeal circulation is required in lung transplantation surgery.     

Einsatz der extrakorporalen Zirkulation (ECLS/ECMO) bei Herz- und Kreislaufversagen

Die Anaesthesiologie - Seit einigen Jahren ist eine stetige Zunahme des Einsatzes von mechanischen extrakorporalen Herz-Kreislauf- und Lungenunterstützungssystemen (ECLS/ECMO) zu verzeichnen....     

Regression of left ventricular hypertrophy following stenting of renal artery stenosis.

PURPOSE: To determine if angioplasty of atherosclerotic renal artery stenosis, which reduces the activation of the renin-angiotensin-aldosterone system (RAAS), may lead to regression of left ventricular hypertrophy. METHODS: The study included 102 patients (58 men; mean age 67 years, range 66-69) who underwent stent-supported percutaneous transluminal renal angioplasty (PTRA) and were included in a clinical follow-up program (mean 24+/-14 months, range 6-60). As a control group, 101 contemporaneous patients (68 men; mean age 68 years, range 66-70) with essential hypertension were investigated. The primary endpoint was the change in left ventricular mass index (LVMI) determined by echocardiography. RESULTS: Mean follow-up intervals were 24+/-14 months (range 6-60) in the study group and 27+/-14 months (range 6-60) in the controls (p = 0.09). LVMI decreased significantly by -10+/-26 g/m(2) in the study group, while it increased significantly by 9+/-28 g/m(2) in the control group (p = 0.001 between groups). In the study group, mean arterial blood pressure was significantly reduced from 99+/-11 mmHg to 90+/-11 mmHg (p<0.0001) during follow-up despite a significant reduction in medication, whereas it increased significantly from 102+/-11 mmHg to 105+/-11 mmHg (p = 0.008) in the control group, although medication was significantly increased. After adjustment for various factors and covariables, PTRA prevailed as an independent predictor for regression of LVMI (p = 0.038). CONCLUSION: PTRA induces regression of LVMI that is independent of the reduction in blood pressure induced by this procedure. Reduced activity of the RAAS may account for this regression.     

Desmoglein-3 is a target autoantigen in spontaneous canine pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease of humans and companion animals. In human patients, PV is associated with the production of IgG autoantibodies specific for keratinocyte desmosomal glycoproteins of the cadherin family. The purpose of this study was to determine whether antikeratinocyte IgG autoantibodies were present in the skin and serum of dogs with PV, and also to identify the canine PV autoantigen(s) targeted by circulating autoantibodies. Eleven dogs were selected because of the microscopic demonstration of suprabasal epithelial acantholysis. Direct immunofluorescence revealed the presence of IgG autoantibodies bound to the membrane of keratinocytes in skin biopsy specimens of 8/9 dogs (89%). Using indirect immunofluorescence, serum-circulating IgG autoantibodies were found in 10/11 (91%) and 5/11 (45%) dogs, using normal canine gingiva and cultured canine oral keratinocytes, respectively. By immunoblotting using cultured canine oral keratinocyte protein lysates, IgG autoantibodies from 7/9 (78%) tested dogs recognized a 130-kDa antigen that comigrated with that identified by rabbit polyclonal antibodies raised against desmoglein-3. This 130 kDa antigen was confirmed to represent the canine equivalent of human desmoglein-3 by immunoprecipitation-immunoblotting. The results of these studies provide evidence that the canine desmoglein-3 homologue is a major autoantigen in dogs with PV. These observations further establish spontaneous canine PV as a natural model for research on pathogenesis, etiology and novel therapeutic approaches for this disease of humans.     

Aortoventriculoplasty: a new technique for the treatment of left ventricular outflow tract obstruction.

Aortoventriculoplasty is a new method of treatment for left ventricular outflow tract obstructions. The concept is based on creating a surgical defect which is patched in such a way as to provide the largest possible outflow to the left ventricle. The incision of the aorta continues down as far as necessary, with the right ventricular wall, the aortic ring, and the septum being cut. Reconstruction with an inner Dacron patch on the septum is completed by replacing the aortic valve with an adequate prosthesis, covering the aortic incision with the same patch, and patching the right venticular opening with an outer patch. This method was used in 4 children with tunnel-like subaortic stenosis, 3 of whom had had unsuccessful previous surgical attempts. Other associated lesions including parachute mitral valve were also corrected during aortoventriculoplasty. Hemodynamic results were excellent following this operation. Two patients died postoperatively, one from advanced myocardial damage and progressive failure and the other from cerebral ischemia caused by insufficient retrograde perfusion through an aortic coarctation that was not repaired earlier. No arrhythmias were observed following the procedure. The other 2 patients are well 7 and 5 months postoperatively with excellent hemodynamic function.     

Results of aortoventriculoplasty in 21 consecutive patients with left ventricular outflow tract obstruction

Results of aortoventriculoplasty (AVP) are reported in 21 patients with various types of left ventricular outflow tract obstruction (LVOTO). The concept of AVP is based on creating a surgical aortoseptal defect which is patched to provide the largest possible outflow tract to the left ventricle. Lesions consisted of isolated diffuse fibromuscular subaortic stenosis in six patients, diffuse subaortic stenosis and associated other cardiovascular anomalies in five, hypoplastic aortic anulus in two, idiopathic hypertrophic subaortic stenosis (IHSS) in two, and stenosis of a previously implanted aortic valvular prosthesis in three patients. Ten patients had had at least one unsuccessful previous surgical attempt to relieve the LVOTO. The coexisting mitral incompetence in IHSS disappeared after AVP alone. Immediate postoperative hemodynamic results were excellent in all cases. Postoperative death in five patients was due to advance myocardial failure in two, brain damage in one, transection of a dominant septal artery in one, and severe acidosis with renal failure in the last case. However, in the last 16 patients (17 operations) the only death (5.8 percent) was that caused by uncontrollable acidosis. Follow-up results indicate that 16 patients are clinically doing well, and hemodynamic studies in 14 patients are rated as excellent or good from 1 to 25 months postoperatively. It is concluded that AVP is an effective operation for managing all types of LVOTO and can be used routinely with an acceptably low mortality rate.