Precise quantification of pressure flow waveforms of a pulsatile ventricular assist device

Unreliable quantification of flow pulsatility has hampered many efforts to assess the importance of pulsatile perfusion. Generation of pulsatile flow depends upon an energy gradient. It is necessary to quantify pressure flow waveforms in terms of hemodynamic energy levels to make a valid comparison between perfusion modes during chronic support. The objective of this study was to quantify pressure flow waveforms in terms of energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE) levels in an adult mock loop using a pulsatile ventricle assist system (VAD). A 70 cc Pierce-Donachy pneumatic pulsatile VAD was used with a Penn State adult mock loop. The pump flow rate was kept constant at 5 L/min with pump rates of 70 and 80 bpm and mean aortic pressures (MAP) of 80, 90, and 100 mm Hg, respectively. Pump flows were adjusted by varying the systolic pressure, systolic duration, and the diastolic vacuum of the pneumatic drive unit. The aortic pressure was adjusted by varying the systemic resistance of the mock loop EEP (mm Hg) = (integral of fpdf)/(integral of fdt) SHE (ergs/cm3) = 1,332 [((integral of fpdt)/(integral of fdt))--MAP] were calculated at each experimental stage. The difference between the EEP and the MAP is the extra energy generated by this device. This difference is approximately 10% in a normal human heart. The EEP levels were 88.3 +/- 0.9 mm Hg, 98.1 +/- 1.3 mm Hg, and 107.4 +/- 1.0 mm Hg with a pump rate of 70 bpm and an aortic pressure of 80 mm Hg, 90 mm Hg, and 100 mm Hg, respectively. Surplus hemodynamic energy in terms of ergs/cm3 was 11,039 +/- 1,236 ergs/cm3, 10,839 +/- 1,659 ergs/cm3, and 9,857 +/- 1,289 ergs/cm3, respectively. The percentage change from the mean aortic pressure to EEP was 10.4 +/- 1.2%, 9.0 +/- 1.4%, and 7.4 +/- 1.0% at the same experimental stages. Similar results were obtained when the pump rate was changed from 70 bpm to 80 bpm. The EEP and SHE formulas are adequate to quantify different levels of pulsatility for direct and meaningful comparisons. This particular pulsatile VAD system produces near physiologic hemodynamic energy levels at each experimental stage.     

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Proliferative response of T lymphocytes to a proline-rich polypeptide (PRP): PRP mimics mitogenic activity of Il-1

Mitogenic properties of a proline-rich polypeptide were investigated. The mitogenic action of PRP was compared with the mitogenic action of Il-1. PRP was not mitogenic for thymocytes at doses 0.01-50 micrograms/ml. PRP, at doses 0.1-50 micrograms/ml, augmented the proliferative response of thymocytes to Con A in a similar fashion as Il-1. At doses higher than 10 micrograms/ml, PRP induced proliferation of lymph node cells and splenocytes as well as T cells from the lymph nodes. It did not, however, cause significant proliferation of B cells from the lymph nodes, at the doses used. PRP did not induce proliferation of an antigen specific Lyt 1+ T cell clone. Il-1 behaved in a similar way as PRP in all the tests described. We consider a possibility that under physiological conditions, at a very early stage of postneonatal life, PRP may replace some functions of Il-1.     

Induction of optimal mucosal antibody responses: effects of age, immunization route(s), and dosing schedule in rats

The antitoxin response in intestinal mucosa was studied in rats immunized either intestinally or by combined parenteral and intestinal dosing with cholera toxin or cholera toxoid. Attention was given to the duration of enteric priming and the magnitude and time course of mucosal anti-cholera toxin responses in rats of defined age. Cholera toxin given only intraduodenally was a more efficient priming immunogen in young rats than in older rats and caused priming that lasted at least 32 weeks; repeated enteric doses increased local priming and repeatedly evoked vigorous mucosal anti-cholera toxin responses which occurred rapidly and declined slowly. Results differed when a portion of the immunizing regimen was parenteral. Cholera toxoid given intraperitoneally (i.p.) caused mucosal priming that peaked promptly and then rapidly declined; parenteral boosting after enteric priming was much more effective given i.p. than subcutaneously; moreover, the booster response was brief, virtually disappearing within 11 days, and could not be reproduced by a second i.p. immunization. These results accord with evidence that parenteral immunization both stimulates and suppresses mucosal secretory immunoglobulin A responses, whereas local immunization is not known to be suppressive. Evidence for parenterally induced suppression was the rapid decline in mucosal priming after i.p. immunization, the shortened mucosal antibody response after i.p. immunization, and possibly the inability to parenterally evoke a booster response twice. In these studies, the level of priming observed at different intervals after parenteral, enteric, or combined immunization appeared to reflect the sum of priming and suppressive effects evoked by the preceding immunization(s).     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Antigen presentation is a function of all B cell subpopulations separated on the basis of size

Purified splenic B cells from nonimmune mice were separated by counterflow centrifugal elutriation into 6 subpopulations containing cells of discrete sizes ranging from 119 to 200 μm³. B cells of each subpopulation were competent to process and present a native globular protein antigen, cytochrome c, to a cytochrome c-specific T cell hybrid. In all cases, the B cells' antigen-presenting function was radiation sensitive and did not require T cells or T cell products, since B cells fixed with paraformaldehyde effectively presented a carboxyl-terminal peptide fragment of cytochrome c containing the T cell determinant. Furthermore, the antigen-presenting function of B cells of each subpopulation was augmented by treatment with submitogenic doses of the F(ab')₂ fragment of rabbit anti-mouse Ig antibodies, in that 10-30-fold fewer B cells were required and higher maximal T cell responses were achieved, indicating that B cells of all sizes are capable of being regulated in their antigen presentation function through their surface Ig. In addition, B cells of each subpopulation responded to soluble factors present in the supernatants of activated T cells as evidenced by an increase in volume and by the uptake of [³H]thymidine. These results indicate that B cells, regardless of size, are able to participate in at least two essential phases of T cell-dependent antibody responses, initiating the interaction by processing and presenting antigen to helper T cells and responding to soluble helper factors secreted by activated T cells.     

Compared colonizing and immunizing efficiency of toxinogenic (A+ B+) Vibrio cholerae and an A- B+ mutant (Texas Star-SR) studied in adult rabbits

Four strains of Vibrio cholerae O1 were compared for their ability to colonize and immunize adult rabbit intestine. Three were virulent, toxinogenic (A+ B+) isolates, and one, an A- B+ mutant (Texas Star-SR), was derived by mutagenesis with nitrosoguanidine. When given orally to nonimmune rabbits, virulent strains colonized the small bowel with similar efficiency, whereas Texas Star-SR colonized poorly. Rabbits fed less than 50 CFU of an A+ B+ strain developed marked serotype-specific resistance to recolonization. In contrast, Texas Star-SR evoked resistance to reinfection less efficiently, with a minimum immunizing dose of 10(5) CFU when given once or 10(3) CFU when given twice. Oral inoculation with an A+ B+ strain also evoked vigorous, dose-dependent mucosal antitoxin responses; comparable inocula of Texas Star-SR were much less effective, causing antitoxin responses that were 90 to 95% smaller. Finally, rabbits inoculated once with 10(4) CFU of an A+ B+ strain were markedly protected against experimental cholera or fecal shedding of V. cholerae when challenged with 10,000 times the 50% effective dose of a virulent strain by the RITARD technique. In contrast, an inoculum of 10(4) CFU of Texas Star-SR was nonprotective, and 10(10) CFU was only partially protective. These studies reveal the remarkable efficiency with which virulent V. cholerae evokes intestinal immunity to recolonization or experimental cholera and show that the A- B+ mutant, Texas Star-SR, is substantially less effective.