Body fat content can be predicted in vivo in mice using a modified dual-energy X-ray absorptiometry technique.

The introduction of transgenic mice as animal models in medical research has increased the need for methods to study the phenotype of mice. The aim of the present study was to develop and evaluate a method for in vivo prediction of fat content in living mice. We combined a modified dual-energy X-ray technique with an image analysis procedure. This combined procedure calculates the percentage of fat area, defined as the percentage of the total area of the mice consisting of >50% fat. A high correlation between the percentage of fat area and dissected adipose tissue was seen in both male and female mice (males, r = 0.92, P < 0.001; females, r = 0.88, P < 0.001). A high correlation was also seen between the percentage of fat area and serum levels of leptin (males, r = 0.95, P < 0.001; females, r = 0.86, P < 0.001). An additional experiment demonstrated a very strong correlation between the percentage of fat area and total body fat as determined by chemical extraction (r = 0.97, P < 0.001). In summary, the percentage of fat area, as measured with the dual-energy X-ray/image combined procedure, provides a good in vivo estimation of total body fat content in mice.

Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth.

Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.

Refinement of, and new applications for, Helicobacter pylori colonization in pig gastric biopsy specimens cultured in vitro.

BACKGROUND: As we have previously reported, pig gastric biopsy specimens cultured in vitro are a highly useful model for studies of Helicobacter pylori growth and adhesion. The aim of this study was to further refine the model in terms of mucosal specificity, culture time, bacterial adhesion, and drug delivery. METHODS: H. pylori-inoculated antral and corporeal pig gastric specimens were cultured for up to 96 h. Biopsy viability, bacterial growth, and adhesion were determined every 24 h. Bismuth subcitrate and omeprazole were added to the top of the specimens via a bio-adhesive gel. RESULTS: Corporeal and antral specimens could be cultured for 72 h and 96 h, respectively, without affecting the viability. In parallel experiments from the same pig the percentage adhesion and total number of adhering H. pylori was higher in corporeal than in antral specimens at 72 h (28% and 4 x 10(5) versus 15% and 4 x 10(4), respectively). Removal of loosely attached H. pylori by rinsing at 24 h doubled the percentage H. pylori adhered during the subsequent 48 h. Bismuth subcitrate had a dose-dependent inhibitory effect on H. pylori; when added to the mucosal side, omeprazole had no effect. CONCLUSION: The pig in vitro biopsy model can be used for detailed H. pylori adhesion studies and for the screening of drugs added to the mucosal or serosal side.

Helicobacter colonization of biopsy specimens cultured in vitro is dependent on both mucosal type and bacterial strain.

BACKGROUND: Colonization by Helicobacter pylori is strictly tissue-specific. We have previously reported on an in vitro adhesion model for pig and human gastric mucosa, in which biopsy specimens were successfully infected and cultured for 72h. The aim of this study was to compare H. pylori colonization of different mucosae and by different Helicobacter strains. METHODS: Specimens from pig, rabbit, and rat antrum, pig urinary bladder, and pig duodenum were inoculated with two H. pylori strains and one H. mustelae strain. Four additional strains, including one mutant lacking flagella, were compared on pig antral specimens. RESULTS: The viability of all mucosae was comparable at 48h of culture. The percentage adhering bacteria increased with time in all mucosae, reaching 17%, 11%, and 2% in pig, rabbit and rat antral mucosa, 11% in pig bladder, and 3% in duodenum at 48h. The type of H. pylori strain was a strong determinant for adhesion in pig antrum. Strain SVA40 had the highest adhesion; the mutant lacking flagella colonized very poorly. H. mustelae adhered to all types of mucosae in a more unspecific manner. CONCLUSIONS: On the basis of tissue viability, bacterial colonization, and adhesion, pig antral mucosa is clearly superior. H. pylori strains differ in their ability to adhere to and colonize cultured mucosa.

Viability, prostaglandin E2 production, and protein handling in normal and inflamed human colonic mucosa cultured for up to 48 h in vitro.

Human colonic mucosa obtained from colon cancer resections ('normal') and from colectomies owing to ulcerative colitis (inflamed) were cultured for up to 48 h in vitro. The 3H-leucine incorporation in normal tissue decreased to 52% (p less than 0.001) at 48 h compared with 24 h. The protein synthesis in normal but not in inflamed explants was significantly (p less than 0.01) improved at 48 h, reaching 72% of the 24-h value, on additions of insulin and the protease inhibitors aprotinin, soyabean trypsin inhibitor, and N alpha-tosyl-L-lysine chloromethyl ketone to the culture medium. Inflamed tissue had significant protein losses of 15% after 24 h and 29% after 48 h in culture, and the excretion of precipitable 3H-leucine-labelled proteins could be as high as 20%/24 h. A slight protein loss was observed in normal tissue after 48 h in culture, but the excretion of labelled proteins was very low (3%). The prostaglandin E2 (PGE2) production in both normal and inflamed tissue displayed an increasing non-linear pattern with time in culture, with higher values for inflamed tissue. The PGE2 release profiles and the differences in basic protein metabolism between normal and inflamed human colonic biopsy specimens in culture might reflect important characteristics of the inflammatory process.

Topographic mapping of Helicobacter pylori colonization in long-term-infected pigs.

Four barrier-born pigs were inoculated with Helicobacter pylori during gastroscopy. Infection in all pigs was established after 3 weeks, and the animals were kept isolated from other pigs in ordinary experimental sites. The pigs were sacrificed and examined 3, 5, 6, and 6.5 months postinoculation. A detailed urease mapping of the pig stomachs showed a patchy distribution of H. pylori. The bacteria colonized in all pigs, with a concentration of H. pylori-positive areas in the antrum and fundus. Furthermore, the number of colonized areas tended to increase with time, and some of these areas showed a strong urease reaction, indicating a heavy colonization with H. pylori. Biopsies from these areas contained 10(2) to 10(5) CFU per 2-mm-wide biopsy. We conclude that persistence of H. pylori infection in barrier-born pigs can be demonstrated for at least 6.5 months. The patchy distribution and the variability of viable bacteria were similar to those described for humans.

Sequential incorporation of 3H- and 14C-arachidonic acid during 24 h in rabbit colonic explants cultured in vitro.

Arachidonic acid (AA) might be released from a number of different intracellular pools. This aspect was studied by sequential incorporation of 3H- and 14C-AA during 24 h in rabbit colonic biopsies in culture. Mucosal explants were subjected to 3H-AA (0-6 h) and 14C-AA (6-24 h) followed by a 2-h washout. Lipid extraction of biopsies revealed a distinct pattern where 3H, in relative terms, dominated in phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and 14C was more abundant in neutral lipids, whereas the remaining lipids had equal proportions of labellings. When stimulating double labelled biopsies with 1 microM phorbol 12-myristate 13-acetate (PMA), 10 micrograms/ml A23187 and a combination thereof, the 14C-labelling was in general released in larger amounts, i.e. a maximum of 7.5% with the combined drugs vs 5% for 3H. However, the relative release of 3H increased upon stimulation especially for PMA + A23187. The release of labelling was directly coupled to a stimulation dependent production of PGE2 in the order PMA less than A23187 less than PMA + A23187, maximum release = 8.2 +/- 2.4 ng/h and 3 biopsies. This study shows the existence of different AA-pools, where PI and PE (3H-labelling) appears to have a slow turnover and representing a terminus in a redistribution chain and where neutral lipids (14C-labelling) have a more rapid turnover. Upon stimulation more AA appears to be recruited from pools with slow turnover.

Adhesion of Helicobacter pylori to human gastric mucosal biopsy specimens cultivated in vitro.

The therapeutic advances in Helicobacter pylori infection is hampered by the lack of suitable animal model systems. We have previously reported on successful adherence of H. pylori to pig gastric mucosa cultured in vitro. The aim of this study was to verify the technique in human biopsy specimens cultured in vitro. Mucosal samples were taken from H. pylori-negative and H. pylori-positive patients undergoing gastric surgery. The non-infected tissue was infected with H. pylori in vitro, and the infected tissue was put into culture immediately. Total number and those H. pylori firmly attached were checked throughout a 72-h culture. Viability of cultured human gastric mucosa was good and unaffected by the presence of H. pylori. The amount of bacteria adhering, increased with time from 0.01% to 2-4% after 72 h in culture. In vivo-infected specimens initially had a low number of firmly attached H. pylori, but total H. pylori increased with time in culture. It is concluded that human gastric biopsy specimens show good viability for 72 h and that viability and cell division of H. pylori were maintained in both in vivo and in vitro H. pylori-infected tissue. In both cases the total number of viable bacteria attached to the specimens increased with incubation time.

Effects of protein synthesis inhibition on PGE2 production in rabbit colonic explants cultured in vitro.

Colonic mucosal biopsies cultured for 6 h in the presence of cycloheximide (CH) showed a dose-dependent inhibition of protein synthesis but a biphasic PGE2 production pattern with an increase in both basal and A23187 stimulated PGE2 release at 0.2 microM. At 10 microM CH both protein synthesis as well as basal and PMA induced PGE2 production was inhibited by 90% whereas A23187 stimulated release showed a 50% decrease. At a dose of 100 microM, CH totally blocked also A23187 stimulated PGE2 release without much further decrease in protein synthesis. The effects of 10 microM CH were time-dependently reversible. In biopsies loaded with 3H-arachidonic acid (AA), 10 microM CH had no apparent effect on phospholipase A2 activity, nor could exogenous AA overcome the CH inhibition of basal PGE2 release. No inhibition of prostaglandin synthetase (PS) activity was found in homogenates of biopsies treated with 10 microM CH for 6 h. No direct effect of CH (up to 1 mM) was seen in control homogenates. It is concluded that at least one step in the PGE2 production is protein synthesis dependent. The effect is however not due to a limitation in the enzymes of the major PS system but more likely to one of its co-factors. This factor only plays a role in the intact cell and its importance seems to be reduced during A23187 conditions possibly due to altered cell status and/or other sources of PS. Commonly used high doses (100 microM) of CH give unspecific effects unrelated to inhibition of protein synthesis.

Endogenous activation of prostaglandin synthesis in rabbit colonic explants cultured in vitro.

Rabbit colonic biopsy specimens cultured in vitro showed an optimal release of prostaglandin E2 (PGE2) after 6-7 h of incubation. The amplitude but not the PGE2 production profile was dependent on stimuli applied--that is, A23187 greater than phorbol 12-myristate 13-acetate greater than control. This study was undertaken to determine the nature of this time-dependent optimum. The following hypotheses were tested: increased substrate availability, de novo synthesis of prostaglandin synthetase (PS), and translocation of PS or increased activity of PS. Arachidonic acid, either continuously present or given as a pulse, increased the overall amplitude equally but did not change the production profile. Cycloheximide, 0.2 microM, which inhibited 50% of the protein synthesis, increased the PGE2 production by 80% at 7 h. The cytoskeletal disrupting agent cytochalasin B had no effect. Homogenates of specimens cultured 6 h showed an increased PGE2 production. This was partly explained by a large increase in the PGE2 production capacity of the particulate matter obtained after 45 min of centrifugation at 100,000 g. It is concluded that the PGE2 production peak is due to an activation of preformed PS and that the PS activity is under the control of a peptide inhibitor, dependent on protein synthesis.

Studies of Helicobacter pylori in a gastric mucosa in vitro animal model.

A gastric mucosa in vitro model for studies of experimental Helicobacter pylori infections has been developed. Biopsy specimens were taken from pig gastric mucosa, infected with H. pylori, and cultured for up to 72 h. To determine the degree of H. pylori adhesion, specimens were vigorously rinsed by vortexing five times before measuring viable count and urease activity. The results showed that it is possible to culture pig gastric mucosa in vitro with maintained viability for at least 72 h. According to the viable count, the bacteria survived and multiplied during the whole culture period. The percentage viable H. pylori in the specimens after rinsing and the urease activity increased with time of culture. The results indicate that the bacteria in the gastric specimens were viable after 72 h and that there was a time-dependent increase in bacterial adhesion to the specimens. This in vitro gastric mucosa model promises to be an applicable and reproducible method, with high capacity, for both pathogenic and mechanistic studies of H. pylori infection.

Culture of normal and inflamed rabbit colonic explants. Medium-term prostaglandin profile.

Rabbit colonic biopsy specimens cultured under well-standardized conditions showed a linear uptake of leucine, thymidine and arachidonic acid (AA) for at least 48 h. Prostaglandin E2 (PGE2) production displayed a time-dependent profile. Initial high and fluctuating levels were seen as a result of the preparation trauma. Within 5-7 h a semi-steady state was reached. Addition of the Ca++ ionophore A23187 significantly increased PGE2 production throughout the 48-h time course, but stimulation was optimal (fivefold) around 6 h, as compared with two- to three-fold at 24 and 48 h. Phorbol 12-myristate 13-acetate stimulation of biopsy specimens labelled for 24 h with 14C-AA showed significantly higher proportional release of 14C-PGE2 than after A23187 or a combination of both, indicating stimulus utilization of different AA pools, with variable turnover. Mucosa from rabbits with acetic acid-induced colitis demonstrated a significantly increased leucine incorporation during the first 24-h period and normal incorporation during the second period. PGE2 production reached a high A23187-insensitive maximum at 24 h. This study shows the importance of sample timing, the existence of different AA pools, and the feasibility of studying inflamed mucosa in medium-term tissue culture.

Induction of and adaptation to olsalazine induced intestinal volume load.

Olsalazine is a new drug for the treatment of ulcerative and Crohn's colitis. The toxicological, pharmacological and pharmacokinetic profiles of olsalazine are excellent; the only side-effect noted from clinical trials with olsalazine is an increased incidence of loose stools and occasional diarrhoea. At high olsalazine concentrations, a direct effect on the net water absorption of the intestine is apparent. This effect is not due to a decrease in absorption, but to an induction of a rapidly reversible, Cl- dependent water secretion. In rats experimentally subjected to olsalazine induced diarrhoea (150 mg/kg), a total adaptation occurs within 3-4 days. This adaptation is not due to tachyphylaxis, but to an increased absorption capacity in the colon and caecum. Similar adaptation has been noted in other species including man.

Pharmacology of olsalazine.

In the treatment of inflammatory bowel disease the main anti-inflammatory component of sulphasalazine is 5-ASA. Based on the same azo-splitting principle in the colon as sulphasalazine, the new drug olsalazine was developed. Olsalazine consists of two 5-ASA molecules joined by an azo bridge. Pharmacokinetic studies have shown that on oral administration there is little systemic absorption of olsalazine. Almost the whole dose passes into the colon, where the olsalazine is completely split into 5-ASA and subsequently excreted in faeces and urine. It can be concluded that from all toxicological and pharmacological aspects, olsalazine is a safe drug. Olsalazine will present the colonic mucosa with twice the amount of 5-ASA/unit compared with sulphasalazine (the present drug of choice) without concomitant delivery of sulphapyridine, which is believed to carry most of the drug's side-effects. Thus, olsalazine seems to be a suitable drug for long-term treatment of ulcerative colitis in man.

Pentagastrin and gastric mucosal blood flow.

The effect of pentagastrin on mucosal microcirculation was studied in rats by use of intravital microscopy. The superficial mucosal vessels were videorecorded for off-line analysis of red cell velocities (VRBC) and vessel diameters, from which blood flow (QRBC) was calculated. Resting mucosal blood flow calculated from single microvascular flow data, and vessel distribution was 40 ml X min-1 X 100 g-1. Pentagastrin infused intravenously in a dose of 20 micrograms X kg-1 X h-1 resulted in submaximal acid secretion (approximately 60%) and a significant increase in QRBC by 47 +/- 14%. When given in a dose of 96 micrograms X kg-1 X h-1 iv, it resulted in maximal acid secretion and an increase in QRBC by 36 +/- 14%. In another series of experiments the results of QRBC measurements during infusion of pentagastrin (20 micrograms X kg-1 X h-1 iv) were compared with those of aminopyrine (AP) clearance or laser-Doppler flowmetry (LDF) in the same animals. Gastric mucosal blood flow determined by [14C]AP clearance increased by 309 +/- 115%, whereas QRBC increased by 34 +/- 11%. When determined by LDF, blood flow increased by 41 +/- 22%, a value similar to the increase in QRBC (50 +/- 19%). Thus, the percent increase in blood flow during pentagastrin infusion estimated by AP clearance was considerably higher than that observed by either direct microvascular measurements or by LDF.

Prostaglandin interaction with histamine release and parietal cell activity in isolated gastric glands.

The effects of different prostanoids on parietal cell activity and glandular histamine (Hi) release were examined in isolated rabbit gastric glands. [14C]aminopyrine accumulation and glandular oxygen consumption were used as indices of parietal cell activity, and Hi was determined fluorophotometrically in the supernatant of the glandular suspensions. Both prostaglandins (PG) E2 and E1 dose dependently (10(-8) and 10(-6) M) increased the release of endogenous Hi. Carbacyclin was less effective and PGF2 alpha was almost without effect. Hi release induced by acetylcholine (Ach) and pentagastrin (Pg) was markedly potentiated in the presence of PGE2 (10(-8) to 10(-5) M). The Ach-induced sti ulation of Hi release was also potentiated by arachidonic acid (10(-5) M), an effect that was inhibitable by the cyclooxygenase inhibitor meclofenamate (3 X 10(-5) M). Somatostatin partially inhibited the response to Pg (3 X 10(-9) M) in combination with PGE2 (10(-5) M). Atropine (10(-5) M) strongly reduced the response elicited by Ach (3 X 10(-6) M) combined with PGE2 (10(-6) M). All prostanoids inhibited Hi (10(-4) M)-induced parietal cell activity in a dose-dependent manner (60-70%) but displayed different potency. The stimulatory response to Ach (3 X 10(-6) M) or Pg (3 X 10(-9) M) in combination with isobutylmethylxanthine (IBMX, 10(-5) M) was inhibited by PGE2 in a dose-dependent fashion. PGE2 (10(-6) M) was considerably more effective than cimetidine (10(-5) M) in inhibiting IBMX (10(-4) M)-stimulated oxygen consumption, and the remaining IBMX-PGE2 response (approximately 40%) was dose dependently (10(-8) to 10(-5) M) inhibited by cimetidine. Addition of Hi (10(-7) to 4 X 10(-7) M) or Pg (3 X 10(-10) to 3 X 10(-9) M) counteracted the PGE2 inhibition of the IBMX response. In addition, IBMX (10(-4) M) combined with PGE2 (10(-6) M) gave rise to a threefold increase in Hi release. These results suggest that prostaglandins have two opposing effects, i.e., liberation of endogenous Hi and inhibition of the action of Hi on the parietal cell.

Electrolyte transport across the basolateral membrane of the parietal cells.

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.

Emerging strategies in ulcer therapy: pumps and receptors.

The treatment of gastric and duodenal ulcers has recently been revolutionised. The reason for this is the increased knowledge of the physiology and biochemistry of the mucosae at the cellular and subcellular levels. In this article, we try to explain how the gastric parietal cell works, and how, based on that knowledge, we might be able to devise increasingly sophisticated tools for inhibition of acid secretion.

The mammalian gastric parietal cell in vitro.

To summarize the metabolic status of the parietal cell: There does not seem to be a close relationship between cellular ATP levels and acid secretion. Acid secretion is absolutely dependent on oxygen, and oxygen consumption will increase in direct proportion to the rate of acid secretion. However, the absolute rate of respiration is not closely related to the formation of acid in the subtissue systems. Acid formation can be driven directly by addition of ATP in permeabilized glands, even under apparent anoxic conditions. This correlates well with the presence of the gastric (H+, K+)-ATPase in the parietal cell. If ATP is the main source of energy for the acid secretion, it is quite possible that the relevant ATP pool is compartmentalized and that the content in this pool has a high turnover rate, whereas the ATP used for other cellular functions would be spared. A pure redox mechanism in the gastric mucosa is not possible. However, it remains to be shown that a redox component is not involved in the secretory process. The acid formation measured by AP accumulation in the gastric glands is not an indication of secretory rate. Thus even though ATP appears to restore acid formation in permeabilized glands, this effect has been mainly studied in nonstimulated systems. A detailed study over the energy requirement in the permeabilized resting cell remains to be done. In the mammals we only have information so far about the piglet and the rabbit in terms of substrate preference. The differences between the two could either be due to species or age difference. In both mammals and amphibia, there is no evidence to suggest that acid secretion results in an increase in oxygen consumption purely due to a state IV to III transition of mitochondrial respiration. Rather, increased Krebs-cycle activity would appear to be the major metabolic result of stimulation.