Liquid jet delivery method featuring S100A1 gene therapy in the rodent model following acute myocardial infarction.

The S100A1 gene is a promising target enhancing contractility and survival post myocardial infarction (MI). Achieving sufficient gene delivery within safety limits is a major translational problem. This proof of concept study evaluates viral mediated S100A1 overexpression featuring a novel liquid jet delivery (LJ) method. Twenty-four rats after successful MI were divided into three groups (n = 8 ea.): saline control (SA); ssAAV9.S100A1 (SS) delivery; and scAAV9.S100A1 (SC) delivery (both 1.2 × 10¹¹ viral particles). For each post MI rat, the LJ device fired three separate 100 µl injections into the myocardium. Following 10 weeks, all rats were evaluated with echocardiography, quantitative PCR (qPCR) and overall S100A1 and CD38 immune protein. At 10 weeks all groups demonstrated a functional decline from baseline, but the S100A1 therapy groups displayed preserved left ventricular function with significantly higher ejection fraction %; SS group (60 ± 3) and SC group (57 ± 4) versus saline (46 ± 3), P < 0.05. Heart qPCR testing showed robust S100A1 in the SS (10,147 ± 3993) and SC (35,155 ± 5808) copies per 100 ng DNA, while off-target liver detection was lower in both SS (40 ± 40), SC (34,841 ± 3164), respectively. Cardiac S100A1 protein expression was (4.3 ± 0.2) and (6.1 ± 0.3) fold higher than controls in the SS and SC groups, respectively, P < 0.05.

Gene delivery technologies for cardiac applications.

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality in the Western society. Advances in understanding the molecular pathology of these diseases, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed these conditions within the reach of gene-based therapy. One of the cornerstones of limiting the effectiveness of gene therapy is the establishment of clinically relevant methods of genetic transfer. Recently there have been advances in direct and transvascular gene delivery methods with the use of new technologies. Current research efforts in IHD are focused primarily on the stimulation of angiogenesis, modify the coronary vascular environment and improve endothelial function with localized gene-eluting catheters and stents. In contrast to standard IHD treatments, gene therapy in HF primarily targets inhibition of apoptosis, reduction in adverse remodeling and increase in contractility through global cardiomyocyte transduction for maximal efficacy. This article will review a variety of gene-transfer strategies in models of coronary artery disease and HF and discuss the relative success of these strategies in improving the efficiency of vector-mediated cardiac gene delivery.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Portal vein thrombosis associated with coronary artery bypass surgery.

Portal vein thrombosis is a rare but well-known complication after abdominal surgery. To our knowledge, there have been no reports about this complication after cardiopulmonary bypass surgery. This can probably be explained by the variety of clinical pictures and difficulties in the establishment of a diagnosis of portal vein thrombosis. Among the possible trigger factors, bacteremia, that is usually caused by Bacteroides fragilis or by Escherichia coli, has been assessed. In this case, several blood culture specimens and fluid from abdominal paracentesis consistently grew coagulase-negative staphylococci which have been proved to be one of the most common pathogens in postoperative infection after cardiac surgery. The patient received clopidogrel before and after coronary artery bypass grafting. We speculate that in this clinical situation associated with coagulase-negative staphylococcal bacteremia, clopidogrel resistance may play an important role. The natural history of portal vein thrombosis in this case is complicated by massive fatal gastrointestinal bleeding from rupture of the esophageal varices. Emergency endoscopic therapy with esophageal variceal ligation, injection of a sclerosing solution and using of vasoconstrictive agents helped only in the first episode of bleeding. Portal vein thrombosis after coronary artery bypass grafting is a serious complication. The clinical picture of portal vein thrombosis may vary greatly and the presence of this condition should be suspected when faced with abdominal pain with gastrointestinal bleeding of unknown origin and sepsis.

Bochdalek Congenital Diaphragmatic Hernia in an Adult Sheep.

Congenital diaphragmatic hernia (CDH) is a rare condition. The aetiology of CDH is often unclear. In our case, a hollow mass was noted on MRI. Cardiac ejection fraction was diminished (47.0%) compared to 60.5% (average of 10 other normal animals, P < 0.05). The final diagnosis of congenital diaphragmatic hernia (Bochdalek type) was made when the sheep underwent surgery. The hernia was right-sided and contained the abomasum. Lung biopsy demonstrated incomplete development with a low number of bronchopulmonary segments and vessels. The likely cause of this hernia was genetic malformation.

Goodpasture's syndrome treated with plasmapheresis. Report of a case.

A 55-year old man had pulmonary hemorrhage and renal insufficiency. Direct immunofluorescence of a renal biopsy specimen disclosed prominent linear deposition of IgA along glomerular capillary walls. The patient underwent clinical remission following plasma exchange and immunosuppressive therapy.

Infant feeding practices of families belonging to a prepaid group practice health care plan.

A survey of the infant feeding practices of 270 families who belonged to a prepaid medical insurance program revealed extensive use of commercially prepared infant formulas during the first months of life, introduction of beikost before 3 months of age and introduction of cow milk into the diet at 3 to 5 months. Ethnic, as well as socioeconomic differences, were observed in the use of milks and formulas, timing of introduction of beikost, and method of feeding solid foods. Average calorie intakes approached or were greater than the recommended dietary allowances. With the exceptions of vitamin D and iron, most infants received much greater than the recommended intake of all nutrients examined. Average sodium intakes were well above advisable intakes. Many infants received supplements of vitamins A, B1, B2, B6, and C in addition to adequate dietary intakes. Results of this survey indicate that many mothers look to pediatricians for advice on how to feed their infants and several findings indicate that nutrition education efforts directed toward pediatricians and parents must be more vigorous than they have been in the past.

Morbid results of prolonged intubation after coronary artery bypass surgery.

OBJECTIVES: This study evaluated the morbid results of prolonged intubation after coronary artery bypass grafting (CABG). METHODS: Over 30 months, 66 of 1,112 patients undergoing CABG required prolonged intubation. They were matched with 66 patients who did not require prolonged intubation. Preoperative and operative variables were evaluated to determine which would predict prolonged intubation. The postoperative courses were then compared to evaluate the effect of prolonged intubation. The study population was divided into three groups: those who underwent early extubation, but required reintubation (n = 24); those who required initial prolonged intubation, but no reintubation (n = 22); and those who required initial prolonged intubation and reintubation (n = 20). RESULTS: Univariate analysis revealed unstable angina (p = 0.037), elevated creatinine (p = 0.001), reduced FEV(1) (p = 0.019), longer cardiopulmonary bypass time (p = 0.009), and a greater positive fluid balance at 24 h (p = 0.0001) as predictors of postoperative prolonged intubation. Multivariate regression analysis revealed elevated creatinine (p = 0.011), FEV(1) (p = 0.022), and fluid balance (p = 0.001) as predictors of prolonged intubation. The study population had longer ICU and hospital stays (p = 0.0001), with more infectious complications (p = 0.0001) and higher mortality (p = 0. 001). In the subgroups of the study population, patients not requiring reintubation had shorter ICU (p = 0.001) and hospital stays (p = 0.0001), fewer infectious complications (p = 0.0001), and reduced mortality (p = 0.0001). CONCLUSIONS: Patients undergoing CABG with reduced FEV(1), renal failure, and positive fluid balance 24 h postoperatively are at risk for prolonged intubation. Prolonged intubation results in significant acute and midterm morbidity and mortality. Early extubation followed by reintubation further increases morbidity and mortality rates in these patients.

Phrenic nerve injury after coronary artery bypass grafting: will it go away?

BACKGROUND: Phrenic nerve injury after coronary artery bypass grafting resolves in most cases. The purpose of this study was to analyze the causes and effects of persistent phrenic nerve injury after coronary artery bypass grafting. METHODS: From a registry of patients with chronic obstructive pulmonary disease who underwent coronary artery bypass grafting, 64 patients were identified who experienced phrenic nerve injury during their operation. Fifteen patients either died during follow-up (n = 9) or were lost to follow-up (n = 6). At the last follow-up visit, all the patients underwent an ultrasound evaluation of the diaphragm and were divided into those who had persistent dysfunction (group I) and those who had normal function (group II). The groups were compared for preoperative and operative risk factors, acute and midterm postoperative results, and quality of life at last follow-up. RESULTS: There were 13 patients in group I and 36 in group II. There were no significant differences in preoperative and operative risk factors between the groups. The length of hospitalization was similar for both groups (9.2 +/- 4.5 versus 8.5 +/- 3.3 days, respectively; p = 0.77). More patients in group I required reintubation (23% versus 14%, respectively; p = 0.04). The mean duration of follow-up was 32.7 +/- 9.2 months. At that time, both groups suffered a reduction of forced expiratory volume in 1 second compared with preoperative values. Group I had a greater reduction in forced expiratory volume in 1 second (p = 0.05). There were a total of 125 postoperative readmissions during the follow-up period, 36 in group I and 89 in group II. There were more admissions because of pulmonary problems in group I (85% versus 53%; p = 0.04). Of the 49 patients, 21 perceived a decline in quality of life after operation. More patients in group I (46% versus 22%; p = 0.05) complained of this decrease. CONCLUSIONS: A significant number of patients who incur phrenic nerve injury after coronary artery bypass grafting have persistent phrenic nerve injury. Patients with persistent phrenic nerve injury have increased acute and midterm morbidity after operation, as well as reduced quality of life.

Interaction of thyroid hormone and heparin in postischemic myocardial recovery.

BACKGROUND: Triiodothyronine (T3) administration can improve postischemic myocardial recovery. Heparin can interfere with cellular binding of T3. Introduction of heparin into an isolated heart model may interfere with this effect. METHODS: Four groups of 8 rat hearts were placed on a modified Langendorff apparatus. All groups underwent 15 minutes of perfusion with modified Krebs-Henseleit solution (KH), followed by 20 minutes of normothermic global ischemia and 30 minutes of reperfusion. Group I underwent reperfusion with KH. Group II underwent reperfusion with KH and 1 x 10(-6) mol/L of T3. In group III, hearts underwent preischemic perfusion with heparinized KH (1,000 U/L) and reperfusion with KH containing 1 x 10(-6) mol/L of T3 and 1,000 U/L of heparin. In group IV, rats were given heparin at 2,000 IU/kg 30 minutes before sacrifice, and isolated hearts were reperfused with KH and 1 x 10(-6) mol/L of T3. A latex balloon in the left ventricle monitored hemodynamic variables. RESULTS: Left ventricular developed pressure throughout postischemic reperfusion was greater in all the groups receiving T3 when compared with group I. Group II showed significantly greater recovery than either group III (p < 0.05) or group IV (p < 0.05). CONCLUSIONS: Addition of T3 to the reperfusate enhances postischemic myocardial recovery in the isolated heart model, whereas addition of heparin reduces this effect.

Phrenic nerve injury after coronary artery grafting: is it always benign?

BACKGROUND: The purpose of this study was to evaluate the effect of phrenic nerve injury (PNI) occurring during coronary artery bypass grafting in patients with major chronic obstructive pulmonary disease (COPD). METHODS: Over a 42-month period, 1,303 patients underwent primary coronary artery bypass grafting. Sixty-seven (5.14%) had major COPD, and 29 (43.3%) of these 67 sustained PNI (group I). These patients were matched for age and ejection fraction with 29 CABG patients with COPD but without PNI (group II), 29 patients without COPD but with PNI (group III), and 29 patients with neither COPD nor PNI (group IV). The groups were compared on the basis of preoperative and operative factors and immediate and midterm morbidity and mortality. RESULTS: There were no significant differences between the groups with respect to hypertension, diabetes, ejection fraction, number of grafts, internal mammary artery use, cardiopulmonary bypass time, and ischemic time. Postoperatively, group I had a longer total hospitalization (group I, 11.7 days; group II, 7.8 days; group III, 7.8 days; and group IV, 6 days; p = 0.0001) and stay in the intensive care unit (I, 3.6 days; II, 2.2 days; III, 2.1 days; and IV, 1.2 days; p = 0.0023). More patients in group I required reintubation (I, 37.9%; II, 3.4%; III, 6.9%; and IV, 0%; p < 0.0001). Mean follow-up was 32.8 months (range, 7 to 48 months). Group I had more hospital readmissions (I, 78; II, 50; III, 61; and IV, 28; p < 0.007) and lower cumulative survival (I, 60.6%; II, 93%; III, 96.8%; and IV, 100%; p < 0.0015) compared with the other groups. CONCLUSIONS: In patients with COPD, PNI during coronary artery bypass grafting has a major negative impact on immediate and midterm results.

Distribution of cerebral flow using retrograde versus antegrade cerebral perfusion.

BACKGROUND: This study compared flow to the brain with retrograde and antegrade cerebral perfusion during circulatory arrest. METHODS: Twenty-four rabbits were injected with 5 mCi of technetium-99 macroaggregated albumin, a tracer trapped in the capillaries. Group I (n = 6) were maintained normothermic, and the tracer was injected into the ascending aorta. Group II (n = 6) were maintained normothermic, and underwent cannulation of the superior vena cava (SVC), exsanguination through the aorta, and injection of the tracer into the SVC, which was proximally occluded. In group III (n = 6), the animal was cooled to 25 degrees C. The animal was exsanguinated through the aorta and tracer was injected into the ascending aorta. In group IV (n = 6), animals were cooled to 25 degrees C. The animal was exsanguinated through the ascending aorta and tracer was injected into the SVC. Three animals (group V) were exsanguinated through the ascending aorta and a retrograde venogram of the SVC was performed. Scintigraphy of groups I to IV was carried out on a digital gamma camera. Brain trapping of tracer was graded from 0 to 5, with 0 being no tracer in the brain and 5 being dominant tracer trapping in the brain. RESULTS: Tracer trapping in the brain showed group I, 3.67+/-0.82; group II, 0; group III, 4.67+/-0.41; group IV, 0.17+/-0.41 (p<0.0001). Retrograde venogram of the SVC showed flow into the cerebral veins. CONCLUSIONS: Retrograde flow through the SVC reaches the cerebral venous system. Flow arriving in retrograde fashion does not go through the capillary system.

Sources of kilocalories and macronutrients in the infant diet.

Sources of kilocalories in the diet of 270 infants from birth to 1 year were examined. Introduction of beikost was rapid: 27 percent of kilocalories in the diet of 2-month-old infants was provided by foods other than milk or formula. Table foods contributed very little to caloric intakes of children less than 5 months of age. Commercially prepared baby foods were the predominant form of beikost given, except to infants in the 9 to 12 months age group. Use of junior foods steadily increased during the first year; by the age of 9 to 12 months, half of the kilocalories from strained and junior foods were provided by junior foods. Proximate composition of the diet was also examined. With increasing age, contributions of protein and carbohydrate to infants' caloric intake were larger and the contribution of fat was lower. Although average percentages of kilocalories from protein, fat, and carbohydrate fell within specified guidelines, proximate composition of the diet of various groups of infants did not conform to these guidelines. These groups included (a) breast-fed infants given beikost, (b) a subgroup of the children fed cow's milk, (c) infants fed skim milk, and (d) children who received a high percentage of kilocalories from beikost.

A needleless liquid jet injection delivery method for cardiac gene therapy: a comparative evaluation versus standard routes of delivery reveals enhanced therapeutic retention and cardiac specific gene expression.

This study evaluates needleless liquid jet method and compares it with three common experimental methods: (1) intramuscular injection (IM), (2) left ventricular intracavitary infusion (LVIC), and (3) LV intracavitary infusion with aortic and pulmonary occlusion (LVIC-OCCL). Two protocols were executed. First (n = 24 rats), retention of dye was evaluated 10 min after delivery in an acute model. The acute study revealed the following: significantly higher dye retention (expressed as % myocardial cross-section area) in the left ventricle in both the liquid jet [52 ± 4] % and LVIC-OCCL [58 ± 3] % groups p < 0.05 compared with IM [31 ± 8] % and LVIC [35 ± 4] %. In the second (n = 16 rats), each animal received adeno-associated virus encoding green fluorescent protein (AAV.EGFP) at a single dose with terminal 6-week endpoint. In the second phase with AAV.EGFP at 6 weeks post-delivery, a similar trend was found with liquid jet [54 ± 5] % and LVIC-OCCL [60 ± 8] % featuring more LV expression as compared with IM [30 ± 9] % and LVIC [23 ± 9] %. The IM and LVIC-OCCL cross sections revealed myocardial fibrosis. With more detailed development in future model studies, needleless liquid jet delivery offers a promising strategy to improve direct myocardial delivery.

Aortic root aneurysm after balloon valvoplasty: evaluation with CT angiography.

A 15-year-old was admitted in 2001 for aortic valve balloon dilatation. He was readmitted in 2005 complaining of fatigue. Echocardiography showed severe aortic regurgitation. Computed tomography revealed aneurysm of aorta near the orifice of the left main coronary artery. He underwent uneventful Ross procedure combined with aneurysmectomy.

Diagnosis of trisomy 21 in preimplantation embryos by single-cell DNA fingerprinting.

Many couples presenting for preimplantation genetic diagnosis (PGD) for a single gene disorder are of advanced reproductive age (>35 years) and have a greater chance of producing embryos with chromosomal aneuploidies. The most common chromosomal aneuploidy observed in newborns is trisomy 21, or Down's syndrome. Consequently, the availability of a highly reliable system that simultaneously detects the heritable gene disorder and trisomy 21 would be beneficial to couples at specific risk. A pentaplex chromosome 21 (Ch 21) single-cell DNA fingerprinting system was developed in a multiplex fluorescence polymerase chain reaction (FL-PCR) on single cells. High reliability and accuracy rates were observed, together with low allele dropout (ADO) and preferential amplification rates on diploid buccal cells, trisomy 21 buccal cells and blastomeres derived from Ch 21 aneuploid embryos. A combined multiplex FL-PCR format was optimized with the common cystic fibrosis delta F508 mutation and validated on single buccal cells from a carrier of the cystic fibrosis delta F508 mutation. This new test is a very powerful technique, which also allows confirmation of the embryo parentage and the identification of extraneous DNA contamination that could cause a misdiagnosis in PGD cases.

Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions.

De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)-PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL-PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL-PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.