Factors affecting interpretation of tissue dielectric constant (TDC) in assessing breast cancer treatment related lymphedema (BCRL).

Tissue dielectric constant (TDC) measurements are increasingly used as quantitative adjunctive tools to detect and assess lymphedema. Various factors affect measured TDC values that may impact clinical interpretations. Our goal was to investigate possible impacts of: 1) anterior vs. medial arm measures, 2) total body water (TBW%) and arm fat percentages (AF%), 3) measurement depth, and 4) skin firmness. In 40 healthy women (24.5±2.5 years), TDC was measured bilaterally on anterior forearm to 0.5, 1.5, 2.5, and 5.0 mm depths using a multiprobe device and on anterior and medial aspects using a compact device. TBW% and AF% were measured at 50KHz and skin firmness measured by skin indentation force (SIF). Results showed: 1) No statistically significant difference in TDC values between anterior and medial arm, 2) a moderate direct correlation between TDC and TBW% (r=0.512, p=0.001), 3) an inverse correlation between TDC and AF% (r= -0.494, p<0.001) with correlations greatest at the deepest depth, and 4) a slight but statistically significant inverse correlation between TDC and SIF (r= -0.354, p=0.001). TDC values with compact vs. multiprobe were within 6% of each other with interarm (dominant/nondominant) ratios not significantly different. The findings provide a framework to help interpret TDC values among divergent conditions.

Stabilization of recombinant adenovirus: site-directed mutagenesis of key asparagine residues in the hexon protein.

The quality and stability of recombinant adenovirus preparations for gene therapy trials are currently analyzed by anion-exchange HPLC. It was shown that the retention time of the adenovirus peak increased over the course of time upon storage in current liquid formulations. The number of isoaspartate residues at the outer surface of the particles also increased in parallel in these preparations. A recombinant adenovirus AV3760 was constructed with a modified hexon protein in which all four accessible Asn residues engaged in Asn-Gly pairs were changed into Leu (Asn244, Asn255, Asn437) or Ala (Asn276). The retention time of AV3760, as measured by HPLC, was unchanged after 2 months at +20 degrees C as opposed to the control adenovirus. This demonstrates that the shift in retention time is caused by an increase in carboxyl groups on the outer surface of the virion due to deamidation of the above Asn residues. The modifications introduced in the hexon protein reduced the rate of Asn deamidation, without adverse effects on the infectivity of the virions. By reducing microheterogeneity in recombinant adenovirus, this work represents a significant advance in the development of a stable pharmaceutical vector for gene therapy.

An improved anion-exchange HPLC method for the detection and purification of adenoviral particles.

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50-74%).

pCOR: a new design of plasmid vectors for nonviral gene therapy.

A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origin of replication requires a plasmid-specific initiator protein, pi protein, encoded by the pir gene limiting its host range to bacterial strains that produce this trans-acting protein; (2) the plasmid's selectable marker is not an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection were constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into murine skeletal muscle.

Degradation and fermentation of alpha-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats.

The ability of several human gut bacteria to break down alpha-1,2 and alpha-1,6 glycosidic linkages in alpha-gluco-oligosaccharides (GOS) was investigated in vitro in substrate utilization tests. Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostridium butyricum, which are usually found in the infant gut and have been associated with both beneficial and deleterious effects on health, were studied. Alpha-Gluco-oligosaccharide degradation was compared in vitro and in vivo in gnotobiotic rats associated with these organisms, inoculated alone or in combination. Oligomer breakdown and short chain fatty acid and gas production indicated hydrolysis and fermentation of the substrate. In vitro and in vivo, Cl. butyricum was the least efficient in utilizing GOS, whereas Bact. thetaiotaomicron was the most efficient. Kinetic studies on GOS hydrolysis in pH-regulated fermenters showed that alpha-1,2 glucosidic bonds, which characterize the substrate, were more resistant than alpha-1,6 linkages. Adaptation of gnotobiotic rats to a diet containing 2% (w/w) GOS significantly increased the hydrolysis of alpha-1,2 glucosidic bonds. Combination of bacteria in trixenic rats improved GOS degradation and inhibited Cl. butyricum metabolism. This inhibition was confirmed in pH-regulated fermenters containing GOS as the principal carbon source. The association of beneficial bacteria and GOS may therefore have a potential health-promoting effect in human neonates.