Determination of periodontal ligament cell viability in the oral rehydration fluid Gatorade and milks of varying fat content

The purpose of this study was twofold: 1) to determine if the oral rehydration fluid Gatorade could serve as a suitable temporary storage medium for maintenance of periodontal ligament (PDL) cell viability on avulsed teeth and 2) to determine if fat content is related to maintenance of cell viability when milk is used as the temporary storage medium. PDL cells were cultured from extracted human teeth then incubated over timed intervals from 15 to 210 min in the oral rehydration fluid Gatorade, milks of varying fat content, and saliva. Dulbeco's Modified Eagles Medium served as the positive control while tap water served as the negative control. Cell viability was determined using a colorimetric assay that used Cell Proliferation Reagent WST-1. Results using Gatorade yielded cell viability data similar to the negative control, tap water, indicating that this oral rehydration fluid was not suitable as a temporary storage medium for the avulsed tooth. However, the fat content of milk was found to have an effect on cell viability, suggesting that milks with lower fat content may be more appropriate for maintaining PDL cell viability than milks with higher fat content.     

Multidisciplinary approach to the repair of vertically fractured teeth

This case report describes the treatment of a vertically fractured upper left second molar. The two segments were extracted separately. The periodontal ligament was protected from damage extraorally by soaking it with Hanks' balanced salt solution. The two segments were bonded with the use of a biocompatible glass ionomer bone cement and replanted in conjunction with an expanded polytetrafluoroethylene (gore-tex) membrane. After 1 yr the tooth functions normally and is clinically and radiographically within normal limits.     

Disease surveillance in the ED: factors leading to the underreporting of gonorrhea

Viable periodontal ligament (PL) cells are required for PL healing of avulsed teeth following replantation. If immediate replantation cannot be accomplished, the ability of PL progenitor cells to reproduce (clonogenic capacity) and recolonize the wound may be extended by prevention of desiccation and storage in physiological media. This investigation examined the effects of storage in saliva, milk, Hank's balanced salt solution (HBSS) and Eagle's medium (alpha MEM) on the clonogenic capacity of human PL progenitor cells at 30 and 60 min extra-alveolar time. Twenty erupted human premolar teeth extracted as atraumatically as possible for orthodontic purposes were used in the present study. Fifteen premolars were placed immediately in freshly collected autologous saliva at room temperature, (+ 23 degrees C) for 15 min. These 15 premolars were next divided into three groups of five and stored in either saliva, milk or HBSS at + 4 degrees C in plastic cups surrounded by ice. The remaining five teeth served as positive controls and were immediately placed in alpha MEM at + 4 degrees C. PL tissue was scraped from one-half of the root surface with a scalpel at 30 and 60 min total extra-alveolar duration. Cells were released from the tissue sample with a 30 min enzymatic digestion procedure and the cells from the tissue samples analyzed for clonogenic capacity. There was a reduction in clonogenic capacity with time for all protocols. Periodontal ligament cells stored in alpha MEM showed the least reduction between 30 and 60 min and the greatest reduction was observed for PL cells stored in saliva. The difference in clonogenic capacity following transfer from saliva to milk or HBSS was not significant at 30 min. At 60 min, cells transferred from saliva to HBSS had a statistically higher percentage of clonogenic cells than those transferred to milk (5.9% vs. 3.5%; P < 0.05). We conclude that immediate storage of avulsed teeth in autologous saliva, followed by transfer to chilled milk, preserves the presence of sufficient progenitor cells in the PL to warrant replantation and the possibility of PL healing at 60 min extra-alveolar duration.     

The osmotic hypersensitivity of the yeast Saccharomyces cerevisiae is strain and growth media dependent: quantitative aspects of the phenomenon

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 M-NaCl; most strains fell within a 300 mM range in LD10 values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mM increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180-1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae.     

The production of interleukin-1 alpha immunoreactivity by human oviductal cells in a coculture system

PURPOSE: The aim of this study was to determine the concentration of interleukin-1 alpha in human embryo culture medium with or without oviductal cell coculture and to correlate the interleukin-1 alpha levels with pregnancy. METHODS: Culture media from 32 in vitro fertilization and embryo transfer cycles were assayed for interleukin-1 alpha by immunoassay technique. Human embryos were cultured in Earles' balanced salt solution supplemented with 15% preovulatory serum (sEBSS) in 16 of these cycles, while embryos in the rest of the cycles were cocultured with human oviductal cells in sEBSS. RESULTS: Both sEBSS and spent sEBSS after embryo culture contained low or undetectable levels of interleukin-1 alpha in the pregnant and nonpregnant cycles. On the other hand, oviductal cells significantly increased the amount of interleukin-1 alpha immunoreactivity in the conventional culture medium or coculture medium (P < 0.001, Mann-Whitney rank sum test). The concentrations of interleukin-1 alpha in the spent sEBSS after oviductal cell culture and after coculture with human embryos were 1.5 +/- 1.0 and 1.3 +/- 0.9 pg/ml, respectively. There was no difference in the interleukin-1 alpha concentration between the pregnant and the nonpregnant coculture cycles. CONCLUSIONS: These data showed that human oviductal cells produced interleukin-1 alpha immunoreactivity in a coculture system. However, this production could not be used as a marker for successful embryo implantation.     

Retention of oral microorganisms on cobalt-chromium alloy and dental acrylic resin with different surface finishes

OBJECTIVES: The purposes of this study were to determine the cytocompatibility of four endodontic sealers and gutta-percha using various extracts and to compare the cellular injuries resulting from these materials in permanent 3T3 monolayers and primary human periodontal ligament fibroblasts. STUDY DESIGN: Set specimens from four sealers (AH26, Apexit, Sealapex, N2) as well as gutta-percha were eluted with cell culture medium for 24 hours, 5 days, 5 days, and 24 hours, respectively. Cytotoxic effects due to these extracts were evaluated by determining proliferation of permanent 3T3 cells and primary human periodontal ligament fibroblasts by means of the fluorochrome propidiumiodide. RESULTS: No statistically significant cellular alterations were induced by gutta-percha and Apexit, whereas various extracts of AH26 and Sealapex caused significant moderate or severe growth inhibition. Severe cellular damage was documented for all extracts of N2, which was statistically significantly different from the other materials in both cell lines (p < 0.05). CONCLUSIONS: Our results confirm that various root canal sealers constantly segregate substances after being exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions and possibly contributing to failure.     

Protective effects of various preservation solutions on cultured endothelial cells

Vascular endothelium represents the first target in organ preservation and plays an important role in reperfusion injury. Bovine aortic endothelial cells were cultivated and the most commonly used preservation solutions, such as University of Wisconsin HTK (Brettschneider's histidine-tryptophane-ketoglutarate), and Euro-Collins solutions were tested on the endothelial monolayer. In addition, one group of cultivated cells was preserved with cold saline solution, and endothelial monolayers grown in culture medium were used as controls. The quality of preservation was assessed after 24, 48, and 72 hours of cold storage. Reperfusion was simulated and its effects were observed by reincubation in culture medium at 37 degrees C for 6 hours. The total number of cells, cell viability (determined using trypan blue exclusion), and morphologic alterations were determined. Prostacyclin release was evaluated as a biochemical marker. University of Wisconsin solution maintains more than 99% cell viability after rewarming after both 24 and 48 hours of cold storage. After 72 hours, 86.7% of cells were still viable. Preservation with HTK and Euro-Collins solution allowed cell survival for only 24 hours (96.7%, HTK; 49.9%, Euro-Collins), with no viable cells seen after 48 hours. The cold saline-preserved sample showed 57.8% viable cells after 24 hours and 29.7% after 48 hours. No viable cells were detectable after 72 hours. Light microscopy revealed several patterns of both structural damage and intracellular change (nucleus and cytoplasm) in the endothelial monolayer after preservation with HTK, Euro-Collins solution, and cold saline solution. No morphologic alterations were seen in the University of Wisconsin solution group for as long as 72 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

The utilization of dipeptides containing L-arginine by chicken macrophages

L-Arginine is the precursor of NO, a cytotoxic agent of macrophages. Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production. A chicken macrophage cell line, the HD11 cell, was used in all experiments. Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM). Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS. Nitrite accumulation in the culture medium was used as the indicator of NO production. At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production. L-Argininyl-L-leucine was 38% as effective as L-arginine. The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM. Both values were slightly higher in a second experiment (97 and 70%, respectively). L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine. In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake. As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.     

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.     

Recovery of nosocomial fecal flora from frozen stool specimens and rectal swabs: comparison of preservatives for epidemiological studies

The recovery of antibiotic-susceptible and -resistant aerobic Gram-negative bacilli from stool specimens and from mock rectal swabs after freezing (-20 degrees C) for as long as 4 weeks was studied using three preservatives: Cary-Blair (CB) transport medium, buffered glycerol saline (BGS), and Para Pak C&S solution (CS). In addition, the recovery of enterococci from rectal swabs was investigated after storage of swabs in Stuart's transport media at 4 degrees C as long as 4 weeks. The log10 decreases in bacterial counts from seeded stool suspensions frozen in BGS were 0.64 (i.e., fourfold) and 1.16 after 1 and 4 weeks, respectively, which were significantly less (p < .05) than 1 and 4 week decreases following freezing in CB (1.57 and 2.85) or in CS (1.50 and 2.45). The recovery of Gram-negative bacilli from patients' rectal swabs preserved in BGS was consistent with the results of the experiments with seeded stool suspensions. There was no detectable decrease in recovery of enterococci from rectal swabs stored at 4 degrees C. BGS performed well as a preservative for freezing stool specimens or rectal swabs for later recovery of nosocomial Gram-negative bacilli; enterococci survived well in refrigerated rectal swab specimens.     

Effect of alveolar lining material on phagocytic and bactericidal activity of lung macrophages against Staphylococcus aureau

Unstimulated rabbits were sacrificed and their lungs washed with heparinized saline. After alveolar macrophages were harvested, the cell-free lavage fluid was centrifuged at 47,000 X g to recover a small, whitish, surface-active pellet (F fraction.) The supernatant was concentrated 15-fold by vacuum dialysis (P fraction). Alveolar macrophages in a serum-free system were challenged with radiolabeled (32P) Staphylococcus aureus preincubated in either balanced salt solution or F or P fraction. A small increase in alveolar macrophage bacterial uptake occurred with P fraction-treated staphylococci. P fraction from locally immunized animals further enhanced phagocytosis. In bactericidial experiments, alveolar macrophages were allowed to phagocytize staphylococci preincubated in either balanced salt solution or F fraction. Intracellular bactericidal activity of alveolar macrophages was quantitated by lysotaphin lysis of extracellular bacteria and quantitation of viable intracellular bacteria. Enhanced lung macrophage bactericidal activity against F fraction-incubated staphylococci was noted.     

Calcium-induced changes on crystallins in organ-cultured porcine lens

In this study, intact porcine lenses were cultured in vitro for 7 days supplemented with commercial balanced salt solution (BSS) which is usually used as an irrigation solution during intraocular surgery, and the lenses were maintained under various culture conditions, e.g. temperature and CO2 concentration. The intact porcine lenses after 7 days culture were analyzed with optical density scanner, gel permeation chromatography on TSK HM-55 column and SDS-PAGE (polyacrylamide gel electrophoresis). It was found that lenses exhibited the least opacity when lenses were cultured with Ca(+2)-free BSS buffer, CO2-free incubator and maintained at a temperature of 25 degrees C. After the lenses were cultured with Ca(+2)-free BSS or BSS medium, the composition of crystallins in lenses was separated with TSK HM-55 column. It was indicated that the percentage of high molecular weight (HMW) protein and (alpha-crystallin increased, and gamma-crystallin decreased in lenses incubated with BSS medium compared with lenses incubated with Ca(+2)-free BSS medium. Following an increase in the concentration of calcium in the medium from 4.3 mM, 20 mM, 50 mM, 100 mM to 200 mM, the opacity of the lens was measured with a densitometer. The changed percentage of various crystallins was similar to lenses with BSS media that increased in HMW protein and alpha-crystallin, decreasing in gamma-crystallin. In the case of lens protein pattern, the crystallin washed from TSK HM-55 gel was separated with SDS-PAGE (polyacrylamide gel electrophoresis). It was indicated that some of proteins disappeared when lenses were incubated with various concentrations of calcium. The vanished pH proteins were 20.5 kDa at 50 mM calcium, 20.5 kDa and 21 kDa at 100 mM, 20.5 kDa, 21 kDa, 22 kDa and 23 kDa at 200 mM which were compared with the protein bands in the presence of 20 mM calcium in BSS medium. This study indicates that the commercial balanced salt solution (BSS) which is usually used as an irrigating solution during intraocular operations may increase the risk for lens opacity because of the calcium contained in the solution.     

Nonlinkage between C6 and chromosome 6 markers

Metacercariae of the trematode Microphalloides japonicus (Osborn, 1919) were cultivated in various media to gravid adults, and the results were compared with those in vivo. A few eggs were produced in the uterus even in balanced salt solution, although cultivation in Eagle's MEM and NCTC 109 media was much better. Addition of heat-inactivated serum resulted in even better development. Encysted metacercariae also produced eggs in some media, but most of them were incomplete and without shells.     

Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsol. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol than in EAS 25 (44.3% +/- 11.7 vs. 25.3% +/- 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by 50% and glutathione levels decreased by approximately 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P < 0.01) in EAS 25 than in Adsol. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.     

Energy metabolism in cochlear outer hair cells in vitro

ATP levels in outer hair cells in vitro were measured using the luciferin/luciferase method. Hair cells were isolated from the guinea pig cochlea and maintained for 2 h in a balanced salt solution with 5.5 mM glucose. Ten to 20 cells sufficed for a robust and reproducible luminescence signal, indicating an ATP content of 6.2 +/- 0.4 fmol/cell. This ATP concentration is similar to that found in cultures of other cell types and agrees well with the classical measurements in freeze-dried preparations. The ATP levels were reduced by the following treatments: (1) the omission of glucose in the culture medium lowered ATP levels by 28%; (2) the inhibition of glycolysis by 2-deoxyglucose lowered ATP levels by 66%; (3) the inhibition of oxidative phosphorylation by carbonyl cyanide m-chlorophenylhydrazine (CCCP) lowered ATP levels by 75%, and (4) the inhibition of both pathways reduced the ATP content to non-detectable levels. Acetoacetate was able to restore ATP levels partially when glycolysis was inhibited. These results suggest that (1) the major pathway of ATP synthesis in outer hair cells is the aerobic metabolism of glucose; (2) endogenous energy stores (e.g. glycogen) can maintain ATP levels in the absence of glucose; and (3) ketone bodies may be alternative energy sources.     

Health personnel and viral hepatitis

The cytotoxicity of restorative dental materials must be investigated to ensure a safe biological response. The MTS assay, a valid and reliable measure of cell viability based on the mitochondrial activity of cultured cells, was used to evaluate the affects on human periodontal ligament (PDL) cells of two resin-modified glass ionomer cements (R-M GICs) (Fuji Duet and Fuji II LC, GC America, Chicago, IL) and one dental amalgam (Contour, Caulk, York, PA)-all suggested materials for root perforation repair. Twelve 4 x 6 mm cylinders of each material were fabricated and placed in 5 ml of alpha-minimum essential medium supplemented with 100 micrograms/ml of penicillin, 50 micrograms/ml of gentamicin, and 5% fetal bovine serum for 24, 48, and 72 h (n = 3). One hundred microliters of eluate was transferred to triplicate wells containing PDL cells previously plated at a density of 10,000 cells/well in a 96-well plate, and incubated for 24 h at 37 degrees C with 5% carbon dioxide. alpha-Minimum essential medium with supplements provided baseline data. Optical density at 490 nm, directly proportional to the number of viable cells, was determined according to manufacturer instructions. Analysis of variance was used to detect differences between treatments and Tukey's HSD (p < 0.05) to detect for differences between group means. Results demonstrated that both material and time affected cell viability (p < 0.0001), with amalgam eluate significantly inhibitory on cell viability at 24 h, compared with control and the two other tested materials. At 48 and 72 h, all three materials exhibited a similar slightly inhibitory effect on the cell viability. Use of resin-modified glass ionomer cement as a root perforation repair material initially (< 24 h) may result in a more favorable response by PDL cells than the tested dental amalgam.     

Isolyte S, a physiologic multielectrolyte solution, is preferable to normal saline to wash cell saver salvaged blood: conclusions from a prospective, randomized study in a canine model

OBJECTIVES: The purpose of this study is to compare normal saline with Isolyte S as the wash solutions during high-volume cell saver autologous blood transfusion. Normal saline, the standard wash solution in cell saver autologous blood transfusion, is associated with acid-base and electrolyte derangements. Isolyte S is a physiologic, balanced multielectrolyte crystalloid solution that approximates the electrolyte content of plasma. DESIGN: Open-label, prospective, randomized study. SETTING: Research laboratory in a Department of Veterans Affairs medical center. SUBJECTS: Fourteen mongrel dogs, weighing 22 to 23 kg each. INTERVENTIONS: Fourteen mongrel dogs were prospectively randomized to receive normal saline (n = 7) or Isolyte S (n = 7). Animals were anesthetized, received heparin for anticoagulation, and underwent 18 cycles of cell saver autotransfusion. In each cycle, 125 mL of blood was arterially withdrawn, and washed with either normal saline (mEq/L) (sodium 154, chloride 154) or Isolyte S (mEq/L) (sodium 141, potassium 5, magnesium 3, chloride 98, phosphate 1, acetate 28, and gluconate 23). The washed blood was retransfused. MEASUREMENTS AND MAIN RESULTS: Acid-base and electrolyte analyses were performed throughout the study on the systemic blood of each group and compared. By the end of the study, the Isolyte S group had a normal pH and an increased bicarbonate concentration (mEq/L: normal values 24 to 32; normal saline 9.0 +/- 1.9 vs. Isolyte S 13.2 +/- 3.0 [p < .01]) and an increased magnesium concentration (mg/dL: normal values 1.6 to 2.4; normal saline 1.6 +/- 0.2 vs. Isolyte S 2.2 +/- 0.2 [p < .0001]). Additionally, the Isolyte S group had a lower chloride concentration (mEq/L: normal values 95 to 110; normal saline 130 +/- 9 vs. Isolyte S 117 +/- 7 [p < .02]) and a lower potassium concentration (mEq/L: normal values 3.5 to 5.0; normal saline 4.4 +/- 0.5 vs. Isolyte S 3.7 +/- 0.3 [p < .01]). There were no significant differences between normal saline or Isolyte S in the values of PCO2, lactic acid, sodium, total and ionized calcium, inorganic phosphorus, total protein, albumin, hemoglobin, and hematocrit. CONCLUSIONS: Fewer systemic acid-base and electrolyte derangements were observed when blood was washed with Isolyte S. Differences between the normal saline and Isolyte S groups are ascribed primarily to the constituents of the wash solution. We conclude that Isolyte S, a physiologic, balanced, multielectrolyte solution, should be considered as the wash solution in high-volume autologous cell saver blood processing and transfusion.     

Apoptosis after intestinal ischemia-reperfusion injury: a morphological study

BACKGROUND: Apoptosis has been identified after ischemia-reperfusion (IR) injury to the brain, heart, kidney, retina, and the adrenals. Intestinal IR injury causes villous and crypt damage, which has so far been attributed to cellular necrosis. This study was undertaken to investigate the possible role of apoptosis after reperfusion of cold-stored small bowel grafts in syngeneic rats. METHODS: Small intestinal grafts were stored at 4 degrees C for 24 hr in saline (n=6) or in modified University of Wisconsin solution (n=6), followed by reperfusion for 1 hr in syngeneic Lewis rats. Small bowel samples were obtained before storage, after preservation and after 1 hr of reperfusion. They were processed for light and electron microscopy and analyzed for cell death, with particular emphasis on apoptosis. RESULTS: Less than one apoptotic event was seen per 10 crypts in normal and stored bowels. An occasional normal and some denuded villous epithelial cells of stored bowels exhibited apoptosis. After isotransplantation and 1 hr of reperfusion, marked increase in apoptosis was seen in the crypts and denuded villous epithelial cells of both saline- and modified University of Wisconsin-stored bowels. Secondary necrosis was seen in apoptotic cells, as were dark cells. Only a few cells showed signs of primary ischemic necrosis. CONCLUSIONS: Apoptosis occurs after intestinal IR injury. Modulation of its genetic regulatory and biochemical effector machinery might alleviate or even prevent IR injury in small bowel transplanted after similar periods of storage.     

Adenosine, inosine, and guanosine protect glial cells during glucose deprivation and mitochondrial inhibition: correlation between protection and ATP preservation

The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 microM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 microM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 microM BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.     

Long-term supplementation of culture medium with essential fatty acids alters alpha-linolenic acid uptake in Caco-2 clone TC7

We investigated the influence of four different culture media: 20% fetal bovine serum (FBS), 5% FBS, 5% FBS supplemented with 10 mg x L(-1) linoleic acid (18:2(n-6)) or alpha-linolenic acid (18:3(n-3)) on alpha-linolenic acid apical uptake in clone TC7 of human intestinal Caco-2 cell line. Neither cellular viability nor cell monolayer integrity and permeability were altered by the four culture conditions. Our results show that the different culture media led to changes in alpha-linolenic acid maximal rate of uptake (Vmax) but did not alter the apparent transport constant (Km). Reducing FBS concentration from 20% to 5% increased significantly the rate of alpha-linolenic acid uptake, which was further increased by supplementation of the medium with 18:2(n-6) or 18:3(n-3). Supplementation with essential fatty acids led to a marked enrichment of brush-border membrane phospholipids in polyunsaturated fatty acids of the corresponding series and decreased significantly the levels of monounsaturated fatty acids. Saturated fatty acids, unsaturation index, and cholesterol/fatty acid ratios were unchanged. No clear relation could be established between the changes in membrane lipid composition and the alterations of alpha-linolenic acid uptake. These results indicate a weak influence of membrane lipid composition in the modulation of the uptake. Therefore, the increase of uptake following long-term supplementation of TC7 cells with essential fatty acids could be attributed to an increase of the expression of membrane protein(s) involved in the apical uptake of long-chain fatty acids. This remains to be established.