Dietary amino acids fed in free form or as protein do differently affect amino acid absorption in a rat everted sac model.

In the present study, the effect of free amino acid (FAA) diets on the intestinal absorption rate of methionine and leucine was studied 'ex vivo' with rats adapted for different periods of time to the diets, using the everted sac method. The adaptation period to the 21% FAA diet with an amino acid content based on casein was either, 0 (no adaptation, N-ADA), 5 (short-term adaptation, ST-ADA), or 26-33 days (long-term adaptation, LT-ADA). Within the ST-ADA and the LT-ADA groups, three different levels of methionine were included: 50%, 100% and 200% of the level normally present in casein. All diets were iso-nitrogenous and iso-caloric. After the adaptation period (0, 5, or 26-33 days), intestinal everted sacs were prepared. Methionine or leucine was added to the medium as transport substrate. The methionine absorption rate of the rats of the LT-ADA groups was higher than that of the N-ADA groups. Furthermore, adaptation to 200% dietary methionine levels caused a significantly slower leucine absorption compared to the 100%, and 50% group. Methionine absorption was similar in the 100% and 200% groups, but the absorption of methionine in the 50% group was enhanced in the distal part of the intestines. We concluded that in response diets with 21% FAAs as only amino acid source, amino acid absorption is decreased to avoid toxic effects of high levels of methionine in the circulation.

Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy.

This is the first report describing recombinant (rec) Newcastle disease virus (NDV) as vector for gene therapy of cancer. The gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF) was inserted as an additional transcription unit at two different positions into the NDV genome. The rec virus with the strongest production of the gene product (rec(GM-CSF)) was selected for our study. The insertion of the new foreign gene did neither affect the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Tumor vaccine cells infected by rec(GM-CSF) stimulated human peripheral blood mononuclear cells (PBMC) to exert antitumor bystander effects in vitro in a tumor neutralization assay. These effects were significantly increased when compared to vaccine infected by rec(-) virus. Furthermore, rec(GM-CSF) led to a much higher interferon-alpha (IFN-alpha) production than rec(-) when added as virus or as virus-modified vaccine to PBMC. Two distinct cell types, monocytes and plasmacytoid dendritic cells were shown to contribute to the augmented IFN-alpha response of PBMC. In conclusion, the already inherent anti-neoplastic and immunostimulatory properties of NDV could be further augmented by the introduction of a therapeutic gene whose product initiates a broad cascade of immunological effects in the microenvironment of the vaccine.

Chiral high-performance liquid chromatography of N-octyl bicycloheptene dicarboximide and confirmatory studies using liquid chromatography-tandem mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy.

N-Octyl bicycloheptene dicarboximide (MGK 264) has exo and endo diastereomers. Each structure has a chiral center at the nitrogen side chain. Enantioselective separation of MGK 264 was achieved by normal-phase high-performance liquid chromatography (HPLC) using cellulose-based Chiralcel OD column with diode-array and optical rotation detectors. Peaks were isolated with the purpose of identifying their stereochemical structures. Molecular mass of the HPLC peaks and their structural information was determined by liquid chromatography-electrospray tandem mass spectrometry (LC-ES-MS-MS). A two-dimensional nuclear magnetic resonance (NMR) spectroscopic technique was used to establish the structural features. Correlation of the data obtained from chiral separation and NMR facilitated in unambiguous assignment of the HPLC peaks.

Significance of D-dimer concentrations during and after cardiopulmonary bypass.

OBJECTIVE: To determine whether D-dimer fragments predictably increase during cardiopulmonary bypass (CPB), and if so, whether increases correlate with postoperative blood loss or predict postoperative coagulopathy. DESIGN: Prospective observational study of 65 consecutive patients undergoing first-time coronary artery bypass graft (CABG) or first-time valve replacement. SETTING: Single center University teaching hospital. PARTICIPANTS: Male and female patients between the ages of 30 and 90 years undergoing first-time CABG or valve replacement surgery using CPB. Patients were excluded from study for prolonged preoperative bleeding time, preoperative warfarin therapy, perioperative intra-aortic balloon pump support, thrombolytic therapy in the week preceding operation, reoperation, and emergency operation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood sampling for platelet count, prothrombin time, partial thromboplastin time, thrombin time, fibrinogen, activated coagulation time (ACT) and D-dimer concentrations was obtained at four times during each case; (1) preoperatively, after insertion of the internal jugular introducer, before insertion of pulmonary artery catheter; (2) during CPB at 28 degrees C, immediately before rewarming; (3) after heparin neutralization (20 minutes after initial protamine dose); (4) 12 to 24 hours postoperatively. Blood loss in the intensive care unit was calculated by measuring total mediastinal drainage output at 1 and 4 hours after arrival from the operating room. An initial decrease in fibrinogen was noted during bypass, but no increase in D-dimer was identified. A few patients developed a modest increase in D-dimer after heparin neutralization, but none greater than 2.0 ug/mL. Postoperatively, fibrinogen concentration increased toward baseline levels. However, this is when six patients developed significant (> 2.0 ug/mL) D-dimer formation. Results suggest appropriate physiologic response-normalization of fibrinogen with new synthesis and remodeling of clot in the operative site causing D-dimer formation. Patients with highest D-dimer levels at 12 to 24 hours postoperatively had the highest blood loss at 4 hours postoperatively, suggesting that early postoperative excess bleeding predisposed to increased clot formation and subsequent clot remodeling causing elevated D-dimer concentrations. CONCLUSIONS: D-dimer concentration is not usually elevated in patients undergoing CPB when adequately anticoagulated as monitored using the ACT. When mild elevation of D-dimer occurs, it is most often after heparin neutralization and/or in the postoperative period and is not predictive of increased postoperative blood loss. Elevations of D-dimer concentrations in the postoperative period without corresponding decreases in fibrinogen concentrations may occur and do not signify coagulopathy.

Detection of proviral DNA and viral RNA in various tissues early after avian leukosis virus infection.

Using molecular biological techniques, a study was made of the tissue tropism of avian leukosis virus (ALV) early after infection. Two strains of chickens, one with and the other without endogenous viral genes, were infected with ALV of subgroup A immediately after hatching; specimens of nine tissues and blood samples were analyzed at various times thereafter. A polymerase chain reaction (PCR), specific to ALV subgroup A, was used to detect proviral DNA and viral RNA. In situ hybridization was used to confirm the presence of proviral DNA in tissue samples and to calibrate the PCR. The pattern of detection of proviral DNA and of ALV-RNA in the various tissues was similar for both chicken strains. At 2 weeks of age, ALV-RNA was demonstrated in all tissues tested: bursa of Fabricius, thymus, bone marrow, proventriculus, liver, spleen, kidney, muscle, gonads, and blood samples, and at 4 weeks of age all tissues contained proviral DNA. No tropism for a specific tissue was observed early after an ALV infection.

Automated real-time data acquisition and analysis of cardiorespiratory function.

Microcomputer generation of an automated record without complexity or operator intervention is desirable in many circumstances. We developed a microcomputer system specifically designed for simplified automated collection of cardiorespiratory data in research and clinical environments. We tested the system during possible extreme clinical conditions by comparison with a patient simulator. Ranges used were heart rate of 35-182 beats per minute, systemic blood pressures of 65-147 mmHg and venous blood pressures of 14-37 mmHg, all with superimposed respiratory variation of 0-24 mmHg. We also tested multiple electrocardiographic dysrhythmias. The results showed that there were no clinically relevant differences in vascular pressures, heart rate, and other variables between computer processed and simulator values. Manually and computer recorded physiological variables were compared to simulator values and the results show that computer values were more accurate. The system was used routinely in 21 animal research experiments over a 4 month period employing a total of 270 collection periods. The file system integrity was tested and found to be satisfactory, even during power failures. Unlike other data collection systems this one (1) requires little or no operator intervention and training, (2) has been rigorously tested for accuracy using a wide variety of extreme patient conditions, (3) has had computer derived values measured against a standardized reference, (4) is reliable against external sources of computer failure, and (5) has screen and printout presentations with quick and easily understandable formats.

Low-flow endobronchial insufflation with air for 2 hours of apnea provides ventilation adequate for survival.

We tested the efficacy of low-flow endobronchial insufflation (EI) with air, as it may be useful in emergency situations in which positive-pressure mechanical ventilation is not possible. The only equipment required is a source of compressed air with flow controller and a specially designed insufflation catheter. The insufflation catheter has forked ends that can be positioned blindly in the mainstem bronchi. In six anesthetized, apneic dogs averaging 9.6 kg, we determined that the average air flow needed to maintain PaO2 above 45 mm Hg and PaCO2 below 65 mm Hg after 30 min was 2.5 L/min. At these flow rates, all dogs survived 2 h of endobronchial insufflation with no neurologic dysfunction. The small internal diameters (0.8 mm) of the catheter ends increase gas exit velocity and allow for adequate oxygenation and CO2 removal at low flow rates. Endobronchial insufflation could also be employed with cricothyroidotomy and a compressor for emergency situations in the field involving mass casualties, where oxygen supplies and equipment are limited.

Preparation of xenon-133 in a lipid emulsion for tissue perfusion studies.

A simple, inexpensive technique for concentrating xenon-133 in a medium suitable for intravenous use is reported. The method uses a 20% lipid emulsion, standard syringes, an oscillating motor, and xenon-133, which is available commercially. Typically, 43% of the xenon-133 combines with the lipid emulsion and remains stable for more than 24 hours in vitro.

Experimental repair of ventricular septal defects using autologous right ventricular muscle flaps: preliminary report.

Survival after repair of postinfarction ventricular septal defects remains poor, often due to extensive loss of contractile muscle in the septum or left ventricle. We evaluated whether a contractile flap of right ventricular muscle could be used to repair a similar ventricular septal defect to augment left ventricular performance in 7 fully instrumented mongrel dogs (weight, 23 to 28 kg). By using hypothermic bypass and cold fibrillatory arrest, a trapezoidal right ventricle flap was fashioned from the free wall of the mid to lower right ventricle, basing its widest portion anteriorly on the septum and left ventricle. A large, 2-cm-diameter core of septum was excised beneath this flap to simulate a postinfarct ventricular septal defect. The right ventricular flap was then invaginated through the defect and sewn to the left ventricular side of the septum with pledgeted sutures taken full thickness through the flap and septum in a "vest-over-pants" fashion. Contraction of the right ventricular flap was confirmed visually and by postbypass multiple gated acquisition scans. The right ventricular defect was closed with fascia lata. All dogs were weaned from bypass without inotropes. Precardiac and postcardiac outputs of 2.5 +/- 0.5 versus 2.3 +/- 0.4 L/min and left ventricular end-diastolic pressures of 4 +/- 2 versus 4 +/- 3 mm Hg were identical. No shunts were detected by oxygen saturation. Autopsies confirmed the integrity of the repair. We conclude that septal defects can be repaired by using contractile right ventricular muscle, thus preserving left ventricular function. This technique offers promise for repair of postinfarction ventricular septal defects by using autologous, already conditioned to contract, cardiac muscle, but its application in humans must await long-term testing.

Collateral ventilation of obstructed lung during high-frequency oscillation in dogs and pigs.

To determine if collateral ventilation (CV) occurs in pigs and dogs during intermittent positive-pressure ventilation (IPPV) and high-frequency oscillation (HFO), seven pigs and seven dogs were studied by measuring Xenon 133 washout (XeW) from an occluded subsegmental bronchus. The rate constant/min (K) for Xe blood uptake (KXeb) was derived, and when subtracted from K for XeW (KXeW) gave K for removal of Xe by CV (KXecv). Pig XeW were single exponentials with mean KXew = 0.25/min during IPPV and 0.12/min with HFO. In pigs, mean K of XeW was no different from KXeb so that all XeW occurred by blood uptake and none by CV. XeW in dogs had two exponentials. Dogs had over 11 times greater mean KXeW than pigs during IPPV and over 24 times greater during HFO. In dogs, on average, 79% (IPPV) and 87% (HFO) of XeW occurred by CV. CV is a means of gas exchange during HFO and IPPV in dogs but not in pigs.

Small bowel obstruction due to disease of epiploic appendage.

Primary disease of an epiploic appendage is rare. Small bowel obstruction is an unusual complication of such disease due to bands or adhesions caused by torsion or inflammation. A 49-year old man presented with small bowel obstruction and was found to have a band from a necrotic epiploic appendage adherent to mesentery, obstructing the ileum. The band was released and the epiploic appendage removed.