Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage.

We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.

Hepatic excretion of cholecystopaques introduced into the canine jejunum.

The biliary excretion of five oral cholecystopaques was measured following their introduction into the jejunum of bile fistula dogs. The mean maximal iodine concentration in the bile ranged form a low of 2.2 +/- 0.55 mg per ml following a dose of 33 mg of iodine per kg of iopanoic acid to a high of 12.9 +/- 1.4 mg per ml after the same dose of sodium ipodate. Cumulative excretion at the end of three hours was over five times greater with sodium ipodate in comparison to ipoanoic acid. It is felt that the best explanation of this date is the ability of the more water soluble media to pass through the unstirred water layer of the intestine.

Radiographic appearance of the nonabsorbed (unconjugated) and conjugated sodium tyropanoate (Bilopaque) in the bowel.

A series of abdominal radiographs were taken in eight normal volunteers after the ingestion of sodium tyropanoate (Bilopaque). These showed nonabsorbed sodium tyropanoate to have a granular appearance, while the conjugated form had a smooth homogeneous appearance. The appearance of conjugated sodium tyropanoate in the bowel has the same diagnostic significance as conjugated iopanoic acid in the presence of a nonvisualized gallbladder. It indicates that the contrast material has passed through the hepatic ductal system and that there has been an opportunity for the gallbladder to opacify.

Factors in the intestinal absorption of oral cholecystopaques.

Interest in the pharmacokinetics of cholecystopaques initially centered on transport from blood to bile. The data obtained in this effort have been valuable and have shown that the maximal iodine concentration achievable in the bile is quite similar for all of the currently available compounds. This concentration is, of course, dose dependent. the transport of contrast material from the bowel to the blood has been shown to be quite variable. Considerable progress was made in understanding this. The tremendous differences in absorption of iopanoic acid depending upon the pH of the administered solution was an initial revelation. The development of the concept that there is a water layer through which the cholecystopaque must pass before reaching the lipid membrane of the intestinal cell has added clarity to understanding the difference in absorption between water-soluble and water-insoluble cholecystopaques. A complete knowledge of what might enhance or inhibit absorption is not known. There is beginning to be an understanding of how intestinal dose relates to plasma levels. This should lead to an optimal dose-timing scheme for each cholecystopaque. The basic assumption is that the highest iodine concentration in the gallbladder leads to the most accurate cholecystography. If this is true, the gallbladder needs to be offered bile at the maximum concentrations during the period preceding filming. To accomplish this, the appropriate plasma level necessary for maximum excretion is needed. Experimental data suggest that our current clinical methods in regard to dose and dose timing need revision to optimize cholecystography. This revision needs to take place with a careful look at toxicity. Accepting the present premise that oral cholecystography can be improved, perhaps without a significant increase in morbidity, a fundamental question to be asked is: is it worth it?

Effect of iopanoate on the biliary and urinary excretion of iodipamide.

The effect of sodium iopanoate and iopanoic acid on the biliary excretion of iodipamide in dogs was studied. Enteric administration of sodium iopanoate within one hour of iodipamide infusion reduced biliary iodipamide excretion and increased urinary iodipamide output. The biliary and urinary excretion of iodipamide was not influenced by iopanoic acid administered 40 and then again 16 hrs before iodipamide. These results suggest that iodipamide cholangiography can be employed 16-18 hrs after a standard two-day iopanoic acid oral cholecystogram without decreasing the ability to visualize the biliary ductal system or increasing the urinary iodipamide excretion.

Validation of a computer-assisted method for estimating the number and volume of gallstones visualized by cholecystography.

It is likely that in the near future there will be widespread use of medicinal therapy to dissolve gallstones. The efficacy of medicinal therapy can best be determined by attempting to relate the total surface area of a collection of gallstones to the composition of bile in patients undergoing therapy. Surface area, in turn, can be directly related to gallstone size and number. In this study, involving 48 cholecystectomized patients, we have shown that standard cholecystography, together with a computer-assisted method of metrology, can effectively monitor the above parameters. Determinations of the standard deviation of 1) replicate readings (35.8%) and 2) averaged metrology estimates compared with actual stone volumes (42.9%), as well as correlation of actual stone volumes with averaged metrology estimates (r = 0.961), indicated the magnitude of assessed change in stone volume that would be necessary to accept a roentgenographic decrease or increase in stone size with 95% confidence. Even with the increased precision found in the computer-assisted method as described, to attain a 98% certainty of some volume change it was necessary to have metrology volume change of 50% or more. Actual stone counts were without significant error in 87.5% of the determinations.

Pyloric and duodenal motor contributions to duodenogastric reflux.

The motor mechanisms of duodenogastric reflux were identified in 4 healthy, conscious dogs using electromyographic and fluoroscopic recordings of stomach, duodenum and upper jejunum. A barium suspension was injected via a pre-placed cannula in the orad jejunum, during the interdigestive period. Under normal conditions, reflux was uncommon. It was produced by duodenal segmental contractions occurring when the pylorus was open, or forced open by the duodenal contractions. Reflux was more common during retrograde electrical pacing of the duodenal pacesetter potential. It was then also produced by duodenal segmental contractions associated with an open, or opening, pylorus. During intravenous administration of apomorphine, reflux occurring early in the vomiting complex, was again produced by duodenal segmental contractions associated with an open, or opening, pylorus. The major apomorphine reflux event, however, occurred later when an emetic antiperistaltic contraction, originating in the duodenum or orad jejunum swept the contents before it into the stomach.

Use of the C-terminal octapeptide of cholecystokinin in clinical radiology. I. The gallbladder.

Two 30-min. infusion doses of the octapeptide of chloecystokinin (C8-CCK) (1 and 4 ng/kg/min) produced equal and consistent gallbladder contractions (75 +/- 8% mean and SEM decrease in size) in 24 human volunteers. The long 30-min infusion method of C8-CCK administration appears better than bolus injection (30 to 180 s) for cholecystokinetic cholecystography.

Use of the C-terminal octapeptide of cholecystokinin in clinical radiology: II. The small bowel.

The octapeptide of cholecystokinin (C8-CCK) administered by bolus (20 ng/kg) and by infusion (4 ng/kg/min for 30 minutes) doses produced significant shortening of small bowel transit time. Significantly, fewer side effects occurred with the infusion method. The problems encountered in studies of small bowel motility are discussed.

Metastatic disease involving the stomach.

Metastatic disease involving the stomach is an unusual and difficult clinical problem. A review of 1010 autopsies of patients with cancer disclosed 17 cases of gastric metastases (an incidence of 1.7%), with breast cancer, lung cancer, and melanoma being the most frequent primaries. The clinical manifestations of epigastric pain, melena, and anemia are nonspecific, necessitating radiographic examination of the gastrointestinal tract. The radiographic findings are usually sufficient to suggest the diagnosis.