Living scaffolds: surgical repair using scaffolds seeded with human adipose-derived stem cells.

BACKGROUND: Decellularized porcine small intestinal submucosa (SIS) is a biological scaffold used surgically for tissue repair. Here, we demonstrate a model of SIS as a scaffold for human adipose-derived stem cells (ASCs) in vitro and apply it in vivo in a rat ventral hernia repair model. STUDY DESIGN: ASCs adherence was examined by confocal microscopy and proliferation rate was measured by growth curves. Multipotency of ASCs seeded onto SIS was tested using adipogenic, chondrogenic, and osteogenic induction media. For in vivo testing, midline abdominal musculofascial and peritoneal defects were created in Sprague-Dawley rats. Samples were evaluated for tensile strength, histopathology and immunohistochemistry. RESULTS: All test groups showed cell adherence and proliferation on SIS. Fibronectin-treated scaffolds retained more cells than those treated with vehicle alone (p < 0.05). Fresh stromal vascular fraction (SVF) pellets containing ASCs were injected onto the SIS scaffold and showed similar results to cultured ASCs. Maintenance of multipotency on SIS was confirmed by lineage-specific markers and dyes. Histopathology revealed neovascularization and cell influx to ASC-seeded SIS samples following animal implantation. ASC-seeded SIS appeared to offer a stronger repair than plain SIS, but these results were not statistically significant. Immunohistochemistry showed continued presence of cells of human origin in ASC-seeded repairs at 1 month postoperation. CONCLUSION: Pretreatment of the scaffold with fibronectin offers a method to increase cell adhesion and delivery. ASCs maintain their immunophenotype and ability to differentiate while on SIS. Seeding freshly isolated SVF onto the scaffold demonstrated that minimally manipulated cells may be useful for perioperative surgical applications within the OR suite. We have shown that this model for a "living mesh" can be successfully used in abdominal wall reconstruction.

Randomized, blinded, placebo-controlled trial of tissue factor pathway inhibitor in porcine septic shock.

This study tested the hypothesis that tissue factor pathway inhibitor (TFPI) would improve mortality and morbidity evoked by peritonitis-induced bacteremia in pigs. Secondarily, it sought to determine if TFPI treatment would attenuate cardiodynamic abnormalities produced by this septic model. 32 pigs were chronically instrumented with intracardiac transducers to measure left ventricular pressure and diameter, pulmonary and aortic pressures, and cardiac output. At least 5 days after surgery to implant transducers, basal cardiovascular readings and blood samples were obtained. Using a randomized, blinded study design, either purified, reconstituted TFPI (1 mg/kg bolus, 10 mg/kg/min for 48 h), placebo (arginine buffer), or saline was administered to pigs immediately after Escherichia coli 0111.B4 (3.0-11 x 10(9) colony-forming U/kg)-laden fibrin clots were implanted intraperitoneally, producing peritonitis and bacteremia. Pigs did not receive antibiotics or supportive therapy. No significant differences in primary or secondary endpoints were noted between the arginine and saline groups, so these data were combined into a control group (N = 20). 5 of 12 TFPI pigs survived (42%), while 5 of 20 control pigs survived (25%); this difference was not significant (p = .714, Fisher's exact test). TFPI treatment augmented cardiac output in surviving pigs, but did not affect any other cardiovascular performance variable (heart rate, % diameter shortening, or systemic and pulmonary vascular resistance). In controls, peritonitis induced rapid increase in plasma tumor necrosis factor-alpha (428 +/- 771 to 5,933 +/- 559 pg/mL at 2 h) and interleukin-8 (180 +/- 153 to 1,393 +/- 145 pg/mL at 2 h). TFPI treatment significantly attenuated cytokine responses to sepsis, reducing peak tumor necrosis factor-alpha to 2,103 +/- 813 pg/mL and reducing peak interleukin-8 levels to 534 +/- 211 pg/mL at 2 h (p < .05, Tukey test, two-way ANOVA). In conclusion, TFPI treatment attenuated important mediator components of the inflammatory response but did not provide significant survival benefit.

A porcine model of peritonitis and bacteremia simulates human septic shock.

Cardiovascular responses to systemic bacteremia were evaluated in a pre-instrumented, conscious pigs. Basal observations were obtained 5-7 days after instrumentation. On the next day, Escherichia coli 0111.B4 (1.1 to 33 x 10(9) CFU/kg)-laden fibrin clots were implanted intraperitoneally. Nonsurvivors (9/18) demonstrated rapid cardiovascular decompensation. Survivors (9/18) demonstrated significant cardiovascular injury, which was reversed by 5-7 days postimplant. Cardiac inotropicity was significantly reduced in this period, but recovered by day 7. Circulating myocardial depressant substance activity (assayed by serum-induced depression of beating neonatal rat myocytes) was present on days 1-4 of bacteremia and recovered to basal values on day 6. No clinical or cardiovascular changes were seen in pigs implanted with sterile clots (n = 4). These data demonstrate that implantation of bacteria-laden fibrin clots in pigs induces cardiovascular alterations that mimic responses seen in human sepsis.

Deleterious effects of buthionine sulfoximine on cardiac function during continuous endotoxemia.

Sepsis has been associated with reversible cardiac injury. To determine whether this injury is mediated by generation of reactive oxidants, tissue glutathione (GSH)--the major intracellular antioxidant--was depleted before endotoxemia. Basal values of cardiac contractile function, perfusion, and cardiac output were measured 5-7 days postsurgery. Salmonella enteritidis endotoxin was continuously infused at 3 micrograms/kg/hr iv via an osmotic pump (Alzet Corp). Endotoxemia significantly reduced myocardial glutathione content (394 +/- 46) to 206 +/- 9 micrograms/g), indicating oxidant stress during endotoxemia. Buthionine sulfoximine (BSO) pretreatment significantly reduced cardiac glutathione in sham pigs from 394 +/- 46 to 199 +/- 26 micrograms/g; and in endotoxemic pigs, BSO pretreatment significantly reduced cardiac glutathione to 106 +/- 18 micrograms/g. Vehicle- and BSO-treated endotoxemic groups demonstrated similar cardiovascular responses to endotoxin challenge. Heart rate increases (122 +/- 15 to 140 +/- 17 bpm) and cardiac outputs decreases (1.50 +/- 0.24 to 1.11 +/- 0.35 l/min) were similar, indicating similar cardiovascular insults induced by endotoxemia. Percent short axis shortening and end-systolic pressure-diameter relation (ESPDR) were significantly reduced in BSO pretreated compared with vehicle-treated endotoxemic pigs. Results support a conclusion that endotoxemia-induced cardiac injury is mediated, in part, by free radical injury. This conclusion is based upon the finding that endogenous myocardial glutathione was depleted by continuous endotoxin infusion and that prior depletion of myocardial glutathione by buthionine sulfoximine exacerbated cardiac injury.

End-systolic elastance as an evaluation of myocardial function in shock.

The response of the heart during sepsis has been studied in human and animal models with disparate results. Because sepsis induces marked peripheral vascular changes, to accurately determine the cardiac response, one must use indices of cardiac performance that are independent of loading conditions and heart rate. The slope of the end-systolic pressure-diameter relationship (ESPDR) has been proposed to have these properties. Pigs were equipped with transducers to measure left ventricular pressure, internal short axis diameter (D), and pulmonary and coronary artery blood flows. After 7-10 days of basal observations, an endotoxin-loaded osmotic pump delivering endotoxin at 10 micrograms/kg/hr was implanted into each pig. Fourteen pigs were so treated, and 4 expired before 24 hr of endotoxin challenge. In the surviving pigs, cardiac output, heart rate, dP/dtmax, and peak systolic pressures were elevated. However, both ESPDR and % D shortening were both significantly depressed. These data suggest that the cardiac response to chronic endotoxin challenge includes a loss of inotropic state as indicated by the load-insensitive indicator, ESPDR, and confirmed by depressed % D shortening. One possible mechanism for reduced inotropic state during endotoxin challenge could be the loss of coronary perfusion. The surviving endotoxin-challenged pigs demonstrated a significant increase in coronary perfusion while stroke work remained unchanged, suggesting that depressed cardiac inotropic state during endotoxin challenge was not caused by reduced coronary blood flow. Rather, the myocardium was relatively overperfused. Another possible mechanism for the loss of cardiac inotropism during endotoxin challenge may be endotoxin-induced generation of reactive oxygen free radicals. This possibility was tested by measuring total cardiac gluthathione, a cellular component depleted by oxidant stress. Endotoxemia reduced these levels 50%. These results suggest that cardiac injury may be mediated by the generation of reactive oxygen free radicals. Further study will determine if this mechanism participates in the loss of cardiac inotropism during endotoxin challenge.

Relation between myocardial glutathione content and extent of ischemia-reperfusion injury.

The relation between the extent of myocardial injury sustained during reperfusion and total glutathione (GSH) content in the ischemic myocardium was examined in anesthetized open-chest pigs subjected to coronary occlusion for 45 minutes and reperfusion for 2 hours. In pigs infused with saline during reperfusion (n = 6) there was a decrease in myocardial GSH content from 380 +/- 48 micrograms/g in normally perfused myocardium to 182 +/- 36 micrograms/g in the ischemic reperfused myocardium (p less than 0.02). Myocardial infarct size (expressed as a percentage of the ischemic area) was 12.5 +/- 0.8%. There was a delay of recovery of contractile function before returning to 60% of preocclusion value. In pigs pretreated with buthionine sulfoximine (BSO) (n = 5), an inhibitor of cellular GSH synthesis, there was reduction in GSH content to 215 +/- 25 micrograms/gm in normally perfused myocardium and to 77 +/- 8 micrograms/gm in the ischemic reperfused myocardium. The extent of injury was greater as evidenced by an increase in infarct size to 30.4 +/- 4.0% (p less than 0.001), severe destructive changes in subepicardial ultrastructure, which were absent in saline-infused pigs, and persistence of dyskinesia throughout reperfusion. In pigs infused with glutathione intravenously (0.8 gm/kg) at a rate of 6.5 mg/kg/min (n = 6), 5 minutes before and continuously during reperfusion, there was an increase in GSH content to 582 +/- 67 micrograms/g in normally perfused myocardium and to 312 +/- 80 micrograms/g in ischemic reperfused myocardium. The increase in myocardial GSH was associated with a reduction in infarct size to 7.5 +/- 1.3% (p less than 0.05, compared with saline-infused pigs) and an early recovery of contractile function of the ischemic myocardium. GSH infusion into pigs pretreated with BSO (n = 4) failed to increase myocardial GSH content and failed to reduce the extent of myocardial injury. Thus, the extent of myocardial injury sustained during reperfusion is very dependent on the effectiveness of its antioxidant defenses. Markedly increased susceptibility to injury occurs when the GSH content in the ischemic myocardium becomes depleted.

Variation in end-systolic pressure-diameter relationship using dP/dtmin or P/Dmax as a definition of end-systole in chronic endotoxemic pigs.

We have reported that cardiac inotropism is reduced in various shock states, most recently during chronic endotoxemia (Lee et al.: American Journal of Physiology 254:H324-H330, 1988) [1]. We based this conclusion upon the alterations observed in the slope of the end-systolic pressure-diameter relationship (ESPDR). Recently, Dietrick and Raymond (Dietrick and Raymond: Surgical Infection Society, 7th Annual Meeting, May, 1987, p 83) [2] have reported that the slope of the end-systolic pressure-wall thickness relationship was augmented in the early stages of sepsis and depressed immediately prior to expiration. One major difference between our studies is the definition of end-systole; we used the time when the ratio of pressure-to-diameter (P/D) in the left ventricle is maximal (P/Dmax), whereas they used the time when the first derivative of pressure is minimal (dP/dtmin). In order to determine if the discrepancy between our conclusions could be explained by the differing definitions of end-systole, data from previous studies were reanalyzed, and the slope of the pressure-diameter relationship at P/Dmax and at dP/dtmin was calculated. Pigs were equipped with instruments to measure left ventricular pressure, short axis diameter, and ECG. Observations during the basal state were obtained 3-7 days after surgery. Chronic endotoxemia was induced by intravenous infusion of S. enteriditis endotoxin via an osmotic minipump at 10 micrograms/kg/hr. During the basal state, the value for the slope of ESPDR at dP/dtmin was lower than the value for the slope of ESPDR at P/Dmax, and there was a good correlation between the two values. During chronic endotoxemia, the slope of ESPDR at dP/dtmin did not change. However, the slope of ESPDR at P/Dmax decreased significantly suggesting that chronic endotoxemia reduced cardiac inotropism. This conclusion is supported by the findings that chronic endotoxemia reduced steady-state values of percentage diameter-shortening (an estimate of ejection fraction) and stable stroke work at significantly higher end-diastolic diameter. These data indicate that it is possible to calculate differing slopes of ESPDR from the same observations dependent upon the time during the cardiac cycle chosen as end-systole. More importantly, these data suggest that during chronic endotoxemia, ventricular relaxation dynamics may change so that postsystolic shortening becomes more prominent and therefore higher values for the slope of ESPDR using pressure and diameter values at dP/dtmin can be calculated.

Electrocardiographic values from clinically normal, anesthetized ferrets (Mustela putorius furo).

Electrocardiograms were recorded from 25 clinically normal male ferrets anesthetized with ketamine and xylazine and from 7 ferrets anesthetized with ketamine alone. Ferrets anesthetized with ketamine alone had excessive salivation, open eyes, muscle tremors, muscle twitching, paddling motions, attempts to stand, and an unstable ECG baseline. Because ketamine/xylazine combination resulted in induction and good muscle relaxation, the ECG had little interference resulting from muscular movement.

Cardiac function and coronary flow in chronic endotoxemic pigs.

We have reported that myocardial inotropism was depressed in acute and chronic endotoxemia. One possible mechanism for this observation is that endotoxemia reduces myocardial perfusion and indeed, we observed reduced myocardial perfusion in acute endotoxemia. This study tested the hypothesis that reduced inotropism of chronic endotoxemia was accompanied by reduced coronary artery blood flow. Fifteen pigs were equipped with left atrial and ventricular catheters, circumflex coronary and pulmonary artery flow meters, left ventricular pressure transducer, and ultrasonic crystals in the anterior-posterior axis to measure internal short axis diameter by sonomicrometry. The pigs recuperated for 3 days before basal data were collected over the next 3-5 days. After at least 7 postoperative days, an osmotic pump containing Salmonella enteriditis endotoxin was implanted in 12 pigs. Endotoxin was delivered at 10 micrograms/hr/kg for 2 days, at which time the animals were sacrificed. Osmotic pumps containing sterile saline were implanted in 3 pigs. Eight of the 12 endotoxemic pigs survived; 4 died before the morning of the second day. The survivors exhibited elevated heart rate, peak left ventricular systolic pressure, and cardiac output. Inotropism was evaluated by calculating the slope of the end-systolic pressure-diameter relationship (ESPDR) and % diameter-shortening. ESPDR was significantly depressed on the second endotoxemic day, while % diameter-shortening was depressed on both endotoxemic days. Coronary artery blood flow was significantly elevated on both endotoxemic days, while cross-sectional stroke work was unchanged. Therefore, the ratio of coronary blood flow to stroke work increased on both endotoxemic days. Nonsurvivors exhibited reduced heart rate, cardiac output, peak left ventricular systolic pressure, ESPDR, and % diameter-shortening. Neither coronary artery blood flow nor flow-to-work ratios increased in this group. Sham endotoxemic pigs demonstrated no cardiac or hemodynamic changes over 3 days. These results indicate that depressed inotropism during chronic endotoxemia was not caused by reduced coronary blood flow; rather, the myocardium was relatively overperfused.

Left ventricular function during chronic endotoxemia in swine.

Cardiac performance was studied in 15 chronically instrumented awake pigs during chronic endotoxemia (CET) induced by intravenous infusion of low doses of endotoxin. We sought to test the hypothesis that left ventricular inotropic state was depressed during the stage of chronic endotoxemia when cardiac output, heart rate, and left ventricular systolic pressures are elevated, termed "hyperdynamic sepsis". Left ventricular pressure, internal short axis diameter (SAX), pulmonary artery blood flow, and electrocardiogram were recorded. After initial surgical preparation, each pig was observed for 7-10 days to measure representative basal values. Each pig was then reoperated on day 10 to implant an endotoxin-loaded osmotic pump whose output, infused Salmonella enteritidis endotoxin at a rate calculated to be 10 micrograms.kg-1.h-1 for up to 7 days. Cardiac performance was monitored by measuring dP/dt, heart rate, stroke volume, end-diastolic diameter, percent change in diameter, and the slope of the end-systolic pressure diameter relationship (ESPDR). Data from the basal days were pooled and compared with the data obtained each day of CET by two-way analysis of variance. Ten of 15 pigs survived more than 2 days of CET; 5 died before the morning of the second CET day. The surviving pigs demonstrated elevated systolic pressures, left ventricular maximum rate of pressure development (+dP/dtmax and -dP/dtmax), heart rates, and cardiac output. However, both ESPDR and percent SAX shortening were significantly depressed during both CET days. We conclude that cardiac inotropic state is depressed during hyperdynamic sepsis as indicated by the load-independent parameter ESPDR and confirmed by depressed percent SAX shortening.

Left ventricular function during lethal and sublethal endotoxemia in swine.

Our previous studies suggested that after a median lethal dose (LD50) of endotoxin, cardiac contractility was depressed in nonsurviving dogs. The canine cardiovascular system is unlike humans in that dogs have a hepatic vein sphincter that is susceptible to adrenergic stimulation capable of raising hepatic and splanchnic venous pressures. We retested the hypothesis that lethality after endotoxin administration is associated with cardiac contractile depression in pigs, because the hepatic circulation in this species is similar to that of humans. We compared cardiac mechanical function of pigs administered a high dose (250 micrograms/kg) or a low dose (100 micrograms/kg) endotoxin by use of the slope of the end-systolic pressure-diameter relationship (ESPDR) as well as other measurements of cardiac performance. In all the pigs administered a high dose, ESPDR demonstrated a marked, time-dependent depression, whereas we observed no significant ESPDR changes after low endotoxin doses. The other cardiodynamic variables were uninterpretable, due to the significant changes in heart rate, end-diastolic diameter (preload), and aortic diastolic pressure (afterload). Plasma myocardial depressant factor activity accumulated in all endotoxin-administered animals, tending to be greater in the high-dose group. In this group, both subendocardial blood flow and global function were depressed, whereas pigs administered the low dose of endotoxin demonstrated slight, but nonsignificant, increases in flow and function. These observations indicate that myocardial contractile depression is associated with a lethal outcome to high doses of endotoxin. One possible mechanism for this loss of contractile function may be a relative hypoperfusion of the subendocardium.

Canine left ventricular function during experimental pancreatitis.

Left ventricular contractility following induction of experimental pancreatitis (EP) was studied. Contractility was evaluated by analyzing the left ventricular end systolic pressure-diameter relationship (sigma ES). Sigma ES is independent of large changes in preload, afterload, and heart rate, but sensitive to changes in ventricular contractility. Following injection of 100,000 IU trypsin in 4% taurocholate into the pancreas to induce EP, seven of eight dogs survived 5 hr. These dogs exhibited an initial significant reduction in mean arterial pressure (MABP) which stabilized at 90% of control at 3-5 hr post-EP. Cardiac output (CO) dropped slowly after EP induction (from 3.08 +/- 0.43 to 2.22 +/- 0.22 liters/min) associated with no significant change in peripheral resistance. Stroke work and stroke volume were markedly depressed reflecting the changes in MABP and CO. No consistent changes in +dP/dt or -dP/dt were observed. The ratio of endo/epicardial blood flow was unchanged as was blood Ca2+ levels throughout the experiment. Ventricular contractility as reflected by sigma ES tended to improve (from 49.7 to 69.6 mm Hg/mm at 4 hr following EP). Therefore, it was concluded that these animals exhibited no loss of ventricular contractility during EP.

Evaluation of ventricular performance in shock.

The behavior of the heart in shock and low flow states is the subject of intense debate in the research and clinical communities. Two major questions concerning cardiac behavior in shock are in need of resolution: Is cardiac contractile function depressed in shock states, and, if so, does cardiac contractile failure initiate cardiovascular collapse? The major orientation of this review is that conclusions based on measurements of the strength of cardiac contraction (parameters dependent on systolic pressure or cardiac output) do not assess cardiac contractility precisely because the strength of cardiac contraction is a function of contractility as well as preload, afterload, and rate. The end-systolic pressure volume relationship (ESPVR) is a parameter that is not a function of rate, preload, or afterload, and significant theoretical and empirical evidence exists to suggest that ESPVR can accurately assess cardiac contractility in vivo. Finally, this article will review reports that have used ESPVR or its approximations to evaluate ventricular contractility in shock models. Several such studies uniformly conclude that cardiac dysfunction is a component of cardiovascular collapse, but whether or not it is an initiating factor is unresolved.

Depression of left ventricular performance during canine splanchnic artery occlusion shock.

We studied canine left ventricular contractile performance following splanchnic artery occlusion (SAO) shock. We evaluated contractile performance by analyzing the left ventricular end systolic pressure-diameter relationship (sigma ES) because we have previously shown that sigma ES is independent of large changes in preload, afterload, and heart rate but sensitive to changes in ventricular contractility. Following release from 2 hours of SAO, seven dogs survived, five expired immediately, and ten expired between 0.5 and 3.5 hours (termed nonsurvivors, [NS]). The NS dogs exhibited slight tachycardia, slight increase in total peripheral resistance, marked decreases in +dP/dt, cardiac output, arterial blood pressure, stroke volume, and stroke work. Ventricular performance (sigma ES) declined with time following SAO release in the nonsurviving dogs; in contrast, surviving animals exhibited an augmentation of sigma ES during SAO and following SAO release. Sham dogs exhibited no time-dependent changes in sigma ES. The dogs that expired immediately following SAO release exhibited a precipitous decline in sigma ES from 43.0 +/- 9.0 to 23.0 +/- 4.8 mm Hg/mm within minutes of SAO release. We analyzed these data by Cox multiple regression analysis to determine the major covariates of survivability. The analysis revealed that sigma ES at the midpoint in time between SAO release and death was best correlated with the survival function. These results suggest that cardiovascular collapse of SAO shock is associated with an early and sustained loss of ventricular contractility.

Canine left ventricular performance during LD50 endotoxemia.

Previous reports of the effect of endotoxin shock on cardiac performance have not achieved uniform results. These discrepancies have possibly been caused by the use of indices of cardiac performance that may have been sensitive to altered heart rate or preconditions of cardiac contraction as well as altered cardiac performance. We tested the hypothesis that, following a median lethal dose (LD50) of E. coli endotoxin, cardiac performance would be diminished in nonsurviving animals and maintained in surviving animals. We elected to employ the analysis of the end-systolic pressure-diameter relationship (sigma ES) as well as other measurements of cardiac performance to test this hypothesis. We established that the sigma ES measurement was independent of increased and decreased afterload and relatively insensitive to altered heart rate. In the nonsurviving animals, sigma ES exhibited a marked depression following endotoxin a administration. In the surviving animals, sigma ES exhibited a nonsignificant decrease followed by a return toward preendotoxin values. All other cardiodynamic measurements were uninterpretable due to the marked changes in heart rate, peripheral vascular function, aortic pressure, and cardiac output. We conclude that, following endotoxin administration, those animals that exhibited a diminished myocardial contractility failed to survive more than 2.5 h postendotoxin, whereas the surviving animals were able to restore normal cardiac contractility. Thus survival of endotoxin administration is associated with the maintenance of normal cardiac contractility.

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis.

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.