Optimization of the annealing temperature for DNA amplification in vitro.

In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.

A remotely programmable insulin delivery system. Successful short-term implantation in man.

A remotely controlled, programmable insulin delivery system was implanted in a diabetic man and the feasibility of the technique was examined. Specific problems included (1) development of an appropriate surgical approach, (2) identification of methods to assess the integrity of the insulin delivery system following implantation, and (3) assessment of plasma glucose and free-insulin profiles obtained with the implanted system. The insulin pump was implanted submuscularly through a midline abdominal incision. The insulin reservoir was placed subcutaneously to allow percutaneous refilling. The insulin delivery catheter terminated in the peritoneal space. No postoperative wound infection occurred and rapid healing of the surgical site ensued. In vivo assessment of the system included (1) dye contrast roentgenography, (2) vasopressin stimulation, and (3) reservoir volume monitoring. Short-acting insulin was then placed in the implanted reservoir and delivered by the system for one month. Mean plasma glucose concentration declined to normal levels, as did glycosylated hemoglobin. Plasma insulin profiles were normalized with appropriate insulin peaks with each meal. We conclude that implantation of a remotely programmable insulin pump is feasible in type I diabetic man. Additional studies are necessary to define which patients will benefit from this type of insulin delivery system.

Five-day programmed intraperitoneal insulin delivery in insulin-dependent diabetic man.

This study examined the feasibility of continuous programmed intraperitoneal insulin infusion to maintain glycemic control in insulin-dependent, "C"-peptide-negative diabetic man over a 5-day period. The insulin was delivered via a transcutaneous ip catheter from a portable, programmable insulin delivery pump. All 10 diabetic subjects received ip insulin during the day and night, and plasma glucose, free insulin, and C-peptide concentrations were evaluated at 16 intervals throughout each 24-h period. Standard American Diabetes Association recommended diets were provided, and the insulin dosage was adjusted for both premeal glycemia and the quantity of calories ingested. All subjects maintained normal daily activities including attendance at work or school but slept in the Clinical Research Center at night. Our results demonstrate that continuous programmed ip insulin infusion can maintain glycemic control in insulin-dependent diabetic man for 5 days. Furthermore, normalization of plasma free insulin profiles can be achieved, with sharp peaks of insulin coincident with the rise in glucose at each meal. We conclude that the peritoneum may be an appropriate insulin delivery site for C-peptide-negative diabetic man.

Normalization of plasma insulin profiles with intraperitoneal insulin infusion in diabetic man.

This study examined the feasibility of normalizing the plasma insulin profile in five insulin deficient diabetic males. Acute meal-related increases in plasma free insulin concentration were achieved by administering short-acting insulin intraperitoneally with a pre-programmed portable rotary splenoid driven pump. This insulin response was compared to that achieved when short-acting insulin was injected subcutaneously 15 minutes prior to each meal. After intraperitoneal insulin maximal plasma free insulin concentration was observed within 45 minutes of administration, and averaged 40 +/- 13 mU/l(+/- SEM) for breakfast, 30 +/- 13 mU/l for lunch, and 36 +/- 13 mU/l for supper. This acute rise was followed by a gradual decline in plasma free insulin concentration, simulating a normal plasma insulin profile. With subcutaneously injected insulin, approximately the same maximal plasma free insulin concentration was obtained as observed with intraperitoneal insulin, but it was delayed 116 minutes following injection. These data suggest that intraperitoneally delivered insulin is rapidly absorbed and may normalize the peripheral plasma free insulin concentration, at least during short-term studies.

Modified hospital pumps for pulsed insulin delivery.

Two standard hospital pumps have been modified to provide bimodal insulin delivery for use as "open loop+ artificial beta cells. The units have been designed to deliver both a low infusion basal rate of insulin for glycemic control during a fasting state in diabetics and a high infusion rate in response to a meal challenge. The basal rate can be varied in steps of 0;2 milliliter per hour from roughly one to three milliliters per hour. The higher infusion rate can be 10 to 20 times the basal rate in steps of two milliliter per hour with an automatic return to the basal rate after a 1- to 99-minute programmable interval. The burst rate is initiated manually at the start of a meal. Displays and monitors are available to indicate the basal and high delivery rates and times. The units have been used for intravenous and intraperitoneal insulin delivery in animals and diabetic patients. There is an improvement in glycemic control and normalization of plasma-free insulin levels in juvenile-onset diabetics treated with this pulsed mode of insulin delivery. The variation in pumping rates provides flexibility in treatment of a variety of glycemic challenges.

Some engineering aspects of insulin delivery systems.

The characteristics of electronically controlled insulin delivery systems are presented. Early experiments with an external system have shown promise in providing improved glycemic control over conventional methods of single or multiple subcutaneous insulin injections. The encouraging results with external insulin delivery systems have led to the development and early testing in dogs of an implantable system with remote controls to permit variable insulin flow rates. A number of questions remain to be answered before widespread experimentation with external and implanted insulin delivery systems is possible. There appears to be no major development problems with the engineering aspects of such systems.

A portable insulin infusion system with a rotary solenoid-driven peristaltic pump.

A portable, external, preprogrammed insulin infusion system with a mass of 560 grams and a battery lifetime of 4 months is described. The system uses a rotary solenoid-driven peristaltic pump, with low-power CMOS timing circuitry and controls to provide bimodal insulin delivery with a timed high rate. Accurate and reproducible delivery of 2 microliter fluid pulses at programmed intervals from 0.6 seconds to 100 minutes can be achieved permitting the delivery of a wide range of insulin concentrations. This system is the precursor to a totally implantable insulin delivery system.