Enteral bile acid treatment improves parenteral nutrition-related liver disease and intestinal mucosal atrophy in neonatal pigs

Total parenteral nutrition (TPN) is essential for patients with impaired gut function but leads to parenteral nutrition-associated liver disease (PNALD). TPN disrupts the normal enterohepatic circulation of bile acids, and we hypothesized that it would decrease intestinal expression of the newly described metabolic hormone fibroblast growth factor-19 (FGF19) and also glucagon-like peptides-1 and -2 (GLP-1 and GLP-2). We tested the effects of restoring bile acids by treating a neonatal piglet PNALD model with chenodeoxycholic acid (CDCA). Neonatal pigs received enteral feeding (EN), TPN, or TPN + CDCA for 14 days, and responses were assessed by serum markers, histology, and levels of key regulatory peptides. Cholestasis and steatosis were demonstrated in the TPN group relative to EN controls by elevated levels of serum total and direct bilirubin and also bile acids and liver triglyceride (TG) content. CDCA treatment improved direct bilirubin levels by almost fourfold compared with the TPN group and also normalized serum bile acids and liver TG. FGF19, GLP-1, and GLP-2 were decreased in plasma of the TPN group compared with the EN group but were all induced by CDCA treatment. Intestinal mucosal growth marked by weight and villus/crypt ratio was significantly reduced in the TPN group compared with the EN group, and CDCA treatment increased both parameters. These results suggest that decreased circulating FGF19 during TPN may contribute to PNALD. Moreover, we show that enteral CDCA not only resolves PNALD but acts as a potent intestinal trophic agent and secretagogue for GLP-2.     

Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease

Home parenteral nutrition (PN) is associated with many complications including severe hepatobiliary dysfunction. Commercial ω-6 fatty acid-soybean based-lipid emulsions in PN may mediate long term PN associate liver disease (PNALD) whereas ω-3-fish oil parenteral emulsions have shown to reverse PNALD in children. However, its clinical effectiveness in adults has been scarcely reported. In this work, we study the role of soybean and fish oil lipid commercial emulsions on inflammatory and profibrotic liver markers in adults with long term PNALD and in in vitro cellular models. Inflammatory and profibrotic markers were measured in serum of ten adults with long term PNALD and in culture supernatants of monocytes. Liver epithelial to mesenchymal transition (EMT) was induced by transforming growth factor beta 1 (TGFβ1) to evaluate in vitro liver fibrosis. Omegaven®, a 100% fish oil commercial emulsion, was infused during four months in two patients with severe long term PNALD reversing, at the first month, the inflammatory, profibrotic and clinical parameters of PNALD. The effect was maintained during the treatment course but impaired when conventional lipid emulsions were reintroduced. The other patients under chronic soybean oil-based PN showed elevated inflammatory and profibrotic parameters. In vitro human monocytes stimulated with lipopolysaccharide induced a strong inflammatory response that was suppressed by Omegaven®, but increased by soybean emulsions. In other experiments, TGFβ1 induced EMT that was suppressed by Omegaven® and enhanced by soybean oil lipid emulsions. Omegaven® improves clinical, anti-inflammatory and anti-fibrotic parameters in adults with long-term home PNALD.     

Total parenteral nutrition adversely affects gut barrier function in neonatal piglets

Sepsis is the most common morbidity in preterm infants, who often receive total parenteral nutrition (TPN). We hypothesized that gut barrier function is compromised in TPN-fed compared with enterally fed newborn piglets (ENT pigs). Colostrum-deprived newborn pigs were implanted with jugular venous and bladder catheters under general anesthesia. Pigs were either administered TPN (n = 15) or fed formula (ENT pigs, n = 15). After 6 days, pigs were gavaged a solution of mannitol, lactulose, and polyethylene glycol 4000 (PEG 4000) and urine was collected for 24 h. At 7 days, small bowel samples were assayed for myeloperoxidase activity, morphometry, and tight junction protein abundance. Intestinal contents and peripheral organ sites were cultured for bacteria. Urinary recovery (%dose) of mannitol (53 vs. 68) was lower, whereas that of lactulose (2.93 vs. 0.18) and PEG 4000 (12.78 vs. 0.96) were higher in TPN vs. ENT pigs, respectively (P < 0.05). Incidence of translocation was similar in TPN and ENT pigs. Myeloperoxidase activity was increased in TPN vs. ENT pigs in the jejunum (P < 0.001) and was weakly correlated with lactulose (R2 = 0.32) and PEG 4000 (R2 = 0.38) recovery. Goblet cell counts did not change, but intraepithelial lymphocyte numbers decreased with TPN. Only claudin-1 protein abundance was increased in the TPN group. We conclude that TPN is associated with impairment of neonatal gut barrier function as measured by permeability but not translocation.     

GLP-2 stimulates intestinal growth in premature TPN-fed pigs by suppressing proteolysis and apoptosis

We wished to determine whether exogenous glucagon-like peptide (GLP)-2 infusion stimulates intestinal growth in parenterally fed immature pigs. Piglets (106-108 days gestation) were given parenteral nutrient infusion (TPN), TPN + human GLP-2 (25 nmol. kg(-1). day(-1)), or sow's milk enterally (ENT) for 6 days. Intestinal protein synthesis was then measured in vivo after a bolus dose of [1-(13)C]phenylalanine, and degradation was calculated from the difference between protein accretion and synthesis. Crypt cell proliferation and apoptosis were measured in situ by 5-bromodeoxyuridine (BrdU) and terminal dUTP nick-end labeling (TUNEL), respectively. Intestinal protein and DNA accretion rates and villus heights were similar in GLP-2 and ENT pigs, and both were higher (P < 0.05) than in TPN pigs. GLP-2 decreased fractional protein degradation rate, whereas ENT increased fractional protein synthesis rate compared with TPN pigs. Percentage of TUNEL-positive cells in GLP-2 and ENT groups was 48 and 64% lower, respectively, than in TPN group (P < 0.05). However, ENT, but not GLP-2, increased percentage of BrdU-positive crypt cells above that in TPN piglets. We conclude that GLP-2 increases intestinal growth in premature, TPN-fed pigs by decreasing proteolysis and apoptosis, whereas enteral nutrition acts via increased protein synthesis and cell proliferation and decreased apoptosis.     

Glucagon-like peptide-2 protects against TPN-induced intestinal hexose malabsorption in enterally refed piglets

Premature infants receiving chronic total parenteral nutrition (TPN) due to feeding intolerance develop intestinal atrophy and reduced nutrient absorption. Although providing the intestinal trophic hormone glucagon-like peptide-2 (GLP-2) during chronic TPN improves intestinal growth and morphology, it is uncertain whether GLP-2 enhances absorptive function. We placed catheters in the carotid artery, jugular and portal veins, duodenum, and a portal vein flow probe in piglets before providing either enteral formula (ENT), TPN or a coinfusion of TPN plus GLP-2 for 6 days. On postoperative day 7, all piglets were fed enterally and digestive functions were evaluated in vivo using dual infusion of enteral ((13)C) and intravenous ((2)H) glucose, in vitro by measuring mucosal lactase activity and rates of apical glucose transport, and by assessing the abundances of sodium glucose transporter-1 (SGLT-1) and glucose transporter-2 (GLUT2). Both ENT and GLP-2 pigs had larger intestine weights, longer villi, and higher lactose digestive capacity and in vivo net glucose and galactose absorption compared with TPN alone. These endpoints were similar in ENT and GLP-2 pigs except for a lower intestinal weight and net glucose absorption in GLP-2 compared with ENT pigs. The enhanced hexose absorption in GLP-2 compared with TPN pigs corresponded with higher lactose digestive and apical glucose transport capacities, increased abundance of SGLT-1, but not GLUT-2, and lower intestinal metabolism of [(13)C]glucose to [(13)C]lactate. Our findings indicate that GLP-2 treatment during chronic TPN maintains intestinal structure and lactose digestive and hexose absorptive capacities, reduces intestinal hexose metabolism, and may facilitate the transition to enteral feeding in TPN-fed infants.     

Qualitative and quantitative comparison of gut bacterial colonization in enterally and parenterally fed neonatal pigs

Total parenteral nutrition (TPN) has been associated with mucosal atrophy, impaired gut barrier function, and translocation of luminal bacteria with resultant sepsis in preterm human infants. Currently, we examined the effects of enteral (ENT) or TPN treatments on translocation events in neonatal pigs and on colonization and composition of microbiota in the neonatal gut. Newborn, colostrum-deprived pigs (<24 hours old) were fitted with intravenous catheters and were fed either ENT (n = 13) or TPN (n = 13) for 7 days. After 7 days of treatment, pigs were euthanized and samples were collected for bacterial culture from the blood, intestinal tract and organs. ENT pigs had increased numbers of bacterial genera isolated, higher concentrations of bacteria (CFU/g), and increased colonization of all segments of the intestinal tract compared to the TPN pigs. Translocation of bacteria from the intestinal tract to tissues or blood was similar (8 of 13) for both groups. The ENT group had 1/13 positive for Clostridium difficile toxin A whereas the TPN group had 5/13. We concluded that ENT favored increased bacterial concentrations comprised of more speciation in the gastrointestinal tract compared to TPN, and that TPN-treated piglets were at higher risk of colonization by toxin-expressing strains of C. difficile.     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Glucagon-like peptide 2 has limited efficacy to increase nutrient absorption in fetal and preterm pigs

Exogenous glucagon-like peptide 2 (GLP-2) prevents intestinal atrophy and increases nutrient absorption in term newborn pigs receiving total parenteral nutrition (TPN). We tested the hypothesis that the immature intestine of fetuses and preterm neonates has a diminished nutrient absorption response to exogenous GLP-2. This was accomplished using catheterized fetal pigs infused for 6 days (87-91% of gestation) with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 7) or saline (n = 7), and cesarean-delivered preterm pigs (92% of gestation) that received TPN with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 8) or saline (n = 7) for 6 days after birth. Responses to GLP-2 were assessed by measuring intestinal dimensions, absorption of nutrients (glucose, leucine, lysine, proline) by intact tissues and brush border membrane vesicles, and abundance of sodium-glucose cotransporter mRNA. Infusion of GLP-2 increased circulating GLP-2 levels in fetuses, but did not increase intestinal mass or absorption of nutrients by intact tissues and brush border membrane vesicles, except for lysine. Administration of exogenous GLP-2 to preterm TPN-fed pigs similarly did not increase rates of nutrient absorption, yet nutrient absorption capacities of the entire small intestine tended to increase (+10-20%, P < 0.10) compared with TPN alone due to increased intestinal mass (+30%, P < 0.05). GLP-2 infusion did not increase sodium-glucose cotransporter-1 mRNA abundance in fetuses or postnatal preterm pigs. Hence, the efficacy of exogenous GLP-2 to improve nutrient absorption by the intestine of fetal and preterm pigs is limited compared with term pigs and more mature animals and humans.     

Change in hepatic and plasma bile acid contents and its regulatory gene expression in the chicken embryo

To investigate changes in bile acid biosynthesis in chicken (Gallus gallus) during embryonic stages, we studied the contribution of hepatic and plasma total bile acid levels, mRNA expression of cholesterol 7 alpha-hydroxylase (CYP7A1), and the expression of its regulatory genes in two embryo models (i.e., broilers and layers) differing in lipid metabolism. Total bile acid levels in plasma and liver were low during embryonic stages, as well as expression of CYP7A1. At hatch (P0), hepatic and plasma total bile acid levels and CYP7A1 mRNA expression in liver were markedly increased in both models. The hepatic mRNA expression of liver X receptor (LXR)alpha, a regulator of CYP7A1 gene expression gradually decreased with developmental stages of both broilers and layers. The hepatic mRNA expression of farnesoid X receptor (FXR), a repressor of CYP7A1 gene expression, also decreased with embryonic development. The present results showed that the mRNA expression of CYP7A1 and synthesis of bile acids was low in embryonic stages, suggesting that FXR might be a key regulator of CYP7A1 gene expression in the chicken embryo.     

Acute alcohol administration inhibits the refeeding response after starvation in rat skeletal muscle

This study determined whether an acute alcohol dose could inhibit the refeeding response in starved muscle. Rats starved for 24 h were pretreated with alcohol or saline before refeeding by intragastric or intravenous infusion of enteral diet (ENT), total parenteral nutrition (TPN), or saline. Refeeding by TPN or ENT stimulated increases in the fractional rate of protein synthesis (k(s)) in skeletal muscle. Alcohol prevented the increase in k(s) when refeeding occurred intragastrically (TPN or ENT) (P < 0.001) but not intravenously (TPN). Upon intragastric refeeding, alcohol inhibited the increase in both eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and p70 S6 kinase (p70(S6K)) phosphorylation in plantaris but caused only partial inhibition in soleus muscle (ENT only). When rats were refed intravenously, alcohol had no effect on the increased 4E-BP1 or p70(S6K) phosphorylation in either muscle. Plasma insulin levels were augmented by alcohol. Alcohol-related changes in plasma amino acid concentrations were similar irrespective of the route of feeding, whereas IGF-I levels showed differential changes. This is the first study to demonstrate that acute alcohol ingestion impedes the starved-to-fed response in skeletal muscle.     

Intravenous lipid composition affects hypoxic pulmonary vasoconstriction in the newborn piglet

To examine the effects of altering the fatty acid (FA) composition of intravenous (IV) lipid emulsions on pulmonary vascular resistance (PVR) and thromboxane production, we studied three groups of newborn piglets after three days of either sow's milk (milk), or total parenteral nutrition (TPN) with either iv soy bean oil (SBO, 52% n-6 and 8% n-3 FA) or fish oil (FO, 5% n-6 and 51% n-3 FA) emulsions. At baseline, and during hypoxia at 20 min and 2 h, cardiac output (Q) was measured, PVR calculated and plasma levels of a prostacyclin metabolite (6-keto-PgF1alpha) and thromboxane B2 (TxB2) were measured. Fatty acid composition of the lung phospholipids was analyzed. There was an exaggerated increase in PVR and decrease in Q during prolonged hypoxia in the TPN-SBO group as compared with the other two groups. There was no difference in PVR and Q between the milk and TPN-FO groups. FA of lung phospholipids reflected the high dietary level of long chain n-3 FA in the TPN-FO group. However, no differences in plasma levels of 6-keto-PgF1alpha or TxB2 were found. Intravenous emulsions made from SBO reduced cardiac output and increased pulmonary vascular resistance in the hypoxic newborn piglet, whereas iv FO emulsions did not. When subjects with pulmonary hypertension are receiving TPN iv SBO may be detrimental; iv FO may be beneficial, giving similar responses as in a milk-fed subject.     

Absence of increase of serum alkaline phosphatase activity with parenteral nutrition-associated cholestasis: possible consequence of hypozincemia and hypomagnesemia

We reviewed charts of 38 Patients who developed cholestasis whilst receiving total parenteral nutrition (TPN). A standard protocol was followed for administration of TPN and included crystalline amino acid solution with lipid emulsion and dextrose as calorie sources. 5 of the 38 patients did not exhibit an increase in alkaline phosphatase. This may be related to the concomitant low levels of either serum zinc or magnesium or both. We also obtained a positive correlation between serum levels of zinc and magnesium and the peak activity of alkaline phosphatase (r = 0.49, p less than 0.01, r-0.55, p less than 0.01, respectively) associated with cholestasis. We hypothesize that the reason for this association is related to both zinc and magnesium being activators of alkaline phosphatase activity.     

Glucagon-like peptide-2 increases sucrase-isomaltase but not caudal-related homeobox protein-2 gene expression

To determine the effect of glucagon-like peptide-2 (GLP-2) on sucrase-isomaltase and caudal-related homeobox protein-2 (Cdx-2) gene expression, male Wistar rats were divided into total parenteral nutrition (TPN)-fed and GLP-2-treated, TPN-fed groups. TPN was given via a jugular line, inserted under anesthesia, for 7 days. The treatment group received 40 microg/day of GLP-2 intravenously with the TPN diet. The small intestine and colon were weighed and measured. Tissue was obtained from the jejunum, terminal ileum, and midcolon. RNA analysis, morphometry, and microdissection were performed. The weight of the small intestine of GLP-2-treated rats was greater than that of TPN-fed rats (P < 0.001). GLP-2 increased the mean metaphase arrests/crypt in both the jejunum and ileum (P < 0.001). Ileal expression of sucrase-isomaltase was increased by 1. 6-fold (P < 0.05). Jejunal expression was increased by a similar amount, although not significantly (P = 0.08). There was no change in Cdx-2 gene expression. Thus GLP-2 can maintain small intestinal morphology and function, but effects on gene expression are not mediated by gross changes in the level of the mRNA for the homeobox protein Cdx-2.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

Supplementary choline attenuates olive oil lipid emulsion‐induced enterocyte apoptosis through suppression of CELF1/AIF pathway

Enterocyte apoptosis induced by lipid emulsions is a key cause of intestinal atrophy under total parenteral nutrition (TPN) support, and our previous work demonstrated that olive oil lipid emulsion (OOLE) could induce enterocyte apoptosis via CUGBP, Elav‐like family member 1 (CELF1)/ apoptosis‐inducing factor (AIF) pathway. As TPN‐associated complications are partially related to choline deficiency, we aimed to address whether choline supplementation could attenuate OOLE‐induced enterocyte apoptosis. Herein we present evidence that supplementary choline exhibits protective effect against OOLE‐induced enterocyte apoptosis both in vivo and in vitro. In a rat model of TPN, substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE. In cultured Caco‐2 cells, supplementary choline attenuated OOLE‐induced apoptosis and mitochondria dysfunction by suppressing CELF1/AIF pathway. Compared to OOLE alone, the expression of CELF1 and AIF was significantly decreased by supplementary choline, whereas the expression of Bcl‐2 was evidently increased. No obvious alterations were observed in Bax expression and caspase‐3 activation. Mechanistically, supplementary choline repressed the expression of CELF1 by increasing the recruitment of CELF1 mRNA to processing bodies, thus resulting in suppression of its protein translation. Taken together, our data suggest that supplementary choline exhibits effective protection against OOLE‐induced enterocyte apoptosis, and thus, it has the potential to be used for the prevention and treatment of TPN‐induced intestinal atrophy.     

Transition from parenteral to enteral nutrition induces immediate diet-dependent gut histological and immunological responses in preterm neonates

Necrotizing enterocolitis (NEC) in preterm infants develops very rapidly from a mild intolerance to enteral feeding into intestinal mucosal hemorrhage, inflammation, and necrosis. We hypothesized that immediate feeding-induced gut responses precede later clinical NEC symptoms in preterm pigs. Fifty-six preterm pigs were fed total parenteral nutrition (TPN) for 48 h followed by enteral feeding for 0, 8, 17, or 34 h with either colostrum (Colos, n = 20) or formula (Form, n = 31). Macroscopic NEC lesions were detected in Form pigs throughout the enteral feeding period (20/31, 65%), whereas most Colos pigs remained protected (1/20, 5%). Just 8 h of formula feeding induced histopathological lesions, as evidenced by capillary stasis and necrosis, epithelial degeneration, edema, and mucosal hemorrhage. These immediate formula-induced changes were paralleled by decreased digestive enzyme activities (lactase and dipeptidylpeptidase IV), increased nutrient fermentation, and altered expression of innate immune defense genes such as interleukins (IL-1α, IL-6, IL-18), nitric oxide synthetase, tight junction proteins (claudins), Toll-like receptors (TLR-4), and TNF-α. In contrast, the first hours of colostrum feeding induced no histopathological lesions, increased maltase activity, and induced changes in gene expressions related to tissue development. Total bacterial density was high after 2 days of parenteral feeding and was not significantly affected by diet (colostrum, formula) or length of enteral feeding (8-34 h), except that a few bacterial groups (Clostridium, Enterococcus, Streptococcus species) increased with time. We conclude that a switch from parenteral to enteral nutrition rapidly induces diet-dependent histopathological, functional, and proinflammatory insults to the immature intestine. Great care is required when introducing enteral feeds to TPN-fed preterm infants, particularly when using formula, because early feeding-induced insults may predispose to NEC lesions that are difficult to revert by later dietary or medical interventions.     

The oil of Adenanthera pavonina L. seeds and its emulsions

The oil of Adenanthera pavonina L. seeds was analysed by chromatographic and instrumental means. The oil was found to be rich in neutral lipids (86.2%), and low in polar lipids (13.8%). The neutral lipids consisted mainly of triacylglycerols (64.2%). Unsaturated fatty acids were found as high as 71%, while the percentage of saturated fatty acids was only 29%. GC and GC/MS analyses revealed linoleic, oleic and lignocerotic acid to be predominant among all fatty acids in the A. pavonina oil, whereas stigmasterol was the major steroid identified within this study. Subsequently, the oil was used for preparation of submicron oil-in-water (o/w) lipid emulsions. Lipid emulsions were formulated by using soybean lecithin (SL) to investigate their particle size, Zeta potential and stability at the different oil and SL ratios. The results obtained indicate possible applications of the tested oil in pharmaceutical and medical fields as drug and cosmetic active ingredient carriers.     

The immunopathological effects of intracolonic injection of Mycobacterium bovis BCG cell wall emulsions in guinea pigs

Summary The immunological and pathological responses of guinea pigs to an intramural colonic injection of emulsions containing cell wall (CW) extracts of Mycobacterium bovis BCG and mineral oil were studied from week 1 to week 36 post-inoculation. The emulsions contained variable concentrations of BCG CW attached to (lipid-phase), or separate from (aqueous-phase), the mineral oil. Delayed cutaneous hypersensitivity to PPD was present throughout the course of the study in a variable percentage of guinea pigs inoculated with either type of emulsion. PPD-induced blast transformation of peripheral blood lymphocytes, studied in guinea pigs which received lipid-phase emulsion, was also detectable throughout the course of the study, with maximal response seen 2 weeks post-inoculation. The intracolonic inoculations were well tolerated, with the exception of the most concentrated lipid-phase emulsion (3 mg/ml BCG CW and 5% oil), after which one of eight guinea pigs died due to a colonic impaction and rupture at the site of inoculation. The pathological response to either type of emulsion was a focal granulomatous colitis, which tended to be more severe as the concentration of BCG CW and oil increased. Extracolonic lesions were usually limited to a granulomatous lymphadenitis of lymph nodes draining the injection site; however, the most concentrated lipid-phase emulsion occasionally produced granulomatous inflammatory foci in the liver and lungs. In general, the lesions induced by the lipid-phase emulsions were more severe than those induced by aqueous-phase emulsions, but the intensity of both types of lesions peaked at 2 or 4 weeks post-inoculation. It was concluded that the guinea pig may serve as a useful model to study BCG immunotherapy of colonic neoplasms, since intracolonic injection of BCG CW resulted in systemic immunity toward mycobacterial antigens and a localized accumulation of macrophages without untoward complications. The abbreviations used in this paper are: BCG CW, bacillus Calmette-Guérin cell walls; PPD, purified protein derivative; PBL, peripheral blood lymphocytes; cpm, counts per minute; SI, stimulation index; DCH, delayed cutaneous hypersensitivity