Characterization of colon carcinoma growth pattern by computerized morphometry: definition of a complexity index.

The invasive front of carcinomas may vary in complexity from smooth to highly complex when the front splits up into small cell clusters or even single cancer cells. The degree of complexity is usually estimated visually and semiquantitatively by a pathologist, although more objective methods based on computer-assisted image analysis are available. In this study, we compared the visual estimation of the irregularity of the tumour invasion front of colon carcinomas to different quantitative image analytical techniques and defined a complexity index for the invasive margin. Sections from 29 archived colon carcinomas were stained immunohistochemically for cytokeratin 8. Images of the tumour invasion front were read into a computer and thresholded so that the tumour tissue became black and the background white or so that the tumour front was outlined by a single pixel line. The invasive front was visually classified into four degrees of irregularity by a pathologist. The complexity of the front was then assessed using four different image analysis techniques, i.e. the estimation of fractal dimension, tumour front length, number of tumour cell clusters and lacunarity. Fractal dimension and tumour cell clusters together gave the best correlation to visual grading using a discriminant analysis. A cluster analysis and a tree diagram analysis were then performed and were found to be superior to visual estimation. The clusters represent different degrees of complexity and the result of the tree diagram analysis can be used to assign complexity indices to colon tumours. The fractal dimension separated tumours up to a certain level (1.5-1.6) of complexity. When the tumour front split up into small cell clusters, the counting of tumour cell clusters separated the cells over and above the fractal dimension. This new technique can be used to objectively and quantitatively describe the complexity of the invasive front of tumours.

Increased liver echogenicity at ultrasound examination reflects degree of steatosis but not of fibrosis in asymptomatic patients with mild/moderate abnormalities of liver transaminases.

AIMS: To investigate whether hyperechogenicity of liver can reliably be interpreted as liver steatosis and if any concomitant or isolated fibrosis can be disclosed. PATIENTS AND METHODS: A series of 165 patients with no signs or symptoms of liver disease referred because of slightly to moderately raised aminotransferases (alanine aminotransferase and/or aspartate aminotransferase 0.7-5.0 microkat/l) for more than 6 months were prospectively investigated with a comprehensive laboratory profile, ultrasound examination of liver and percutaneous liver biopsy Fibrosis was assessed quantitatively and according to Metavir. Steatosis was graded as none, mild, moderate or severe. RESULTS: Of 98 (59.4%) patients with raised echogenicity, 85 (86.7%) had liver steatosis of at least moderate degree, 9 patients with same degree of steatosis had normal echogenicity and 13 patients with no or only mild steatosis had a hyperechogenic liver (sensitivity 0.90, specificity 0.82, positive predictive value 0.87, negative predictive value 0.87). About the same relations were found regardless of body mass index and degree of fibrosis. With increased echogenicity together with high attenuation (n = 591 and reduced portal vessel wall distinction (n = 79), positive predictive value increased to 0.93 and 0.94, respectively. Quantitatively assessed fibrosis (mean +/- SD) was 3.2 +/- 4.6% of biopsy area with normal and 2.3 +/- 1.8% with raised echogenicity (ns). Echogenicity was normal in 5 out of 9 patients with septal fibrosis and in 4 out of 6 patients with cirrhosis. Any structural, non-homogenous findings at ultrasound were not associated with architectural fibrotic changes and none had nodular contours of liver surface. CONCLUSIONS: Assessment of liver echogenicity is of value for detection or exclusion of moderate to pronounced fatty infiltration (correct classification 86.6%) but cannot be relied upon in diagnosing fibrosis, not even cirrhosis in asymptomatic patients with mild to moderately elevated liver transaminases.

Prevalence of subtypes of intestinal metaplasia in the general population and in patients with autoimmune chronic atrophic gastritis.

BACKGROUND: Gastric intestinal metaplasia (IM) is seen mostly in association with chronic gastritis, induced either by Helicobacter pylori infection or autoimmune mechanisms. IM can be categorized into three subtypes, where type III is associated with gastric carcinoma of intestinal type. METHODS: Gastric biopsies from 475 subjects randomly selected from the general population and from 27 patients with autoimmune gastritis associated with pernicious anaemia were used. The criteria of Filipe & Jass were applied using different histochemical techniques in combination with haematoxylin and eosin stained material. RESULTS: Twenty-three percent (109/475) of the subjects from the general population and 88% (24/27) in the group with autoimmune gastritis had IM. Type III IM occurred in 4% in both populations. Type III IM was located in the antrum in 90% in the general population. In the group with autoimmune gastritis, only one patient had type III IM, which was located in the corpus. CONCLUSIONS: This study reveals for the first time the prevalence and distribution of subtypes of IM in a general population from the Western world. The comparatively high prevalence of type III IM in the general population (4%) indicates that its role as a precursor of gastric carcinoma may have been overemphasized. A similar prevalence of type III IM in patients with autoimmune gastritis may be considered low and suggests that mechanisms for gastric carcinogenesis other than the atrophy-metaplasia-dysplasia sequence could also operate in this condition.

Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease.

BACKGROUND: Crohn's disease is associated with deranged intestinal permeability in vivo, suggesting dysfunction of tight junctions. The luminal contents are important for development of neoinflammation following resection. Regulation of tight junctions by luminal factors has not previously been studied in Crohn's disease. AIMS: The aim of the study was to investigate the effects of a luminal stimulus, known to affect tight junctions, on the distal ileum in patients with Crohn's disease. PATIENTS: Surgical specimens from the distal ileum of patients with Crohn's disease (n=12) were studied, and ileal specimens from colon cancer patients (n=13) served as controls. METHODS: Mucosal permeability to 51Cr-EDTA and electrical resistance were studied in Ussing chambers during luminal exposure to sodium caprate (a constituent of milk fat, affecting tight junctions) or to buffer only. The mechanisms involved were studied by mucosal ATP levels, and by electron and confocal microscopy. RESULTS: Baseline permeability was the same in non-inflamed ileum of Crohn's disease and controls. Sodium caprate induced a rapid increase in paracellular permeability--that is, increased permeation of 51Cr-EDTA and decreased electrical resistance--which was more pronounced in non-inflamed ileum of Crohn's disease, and electron microscopy showed dilatations within the tight junctions. Moreover, sodium caprate induced disassembly of perijunctional filamentous actin was more pronounced in Crohn's disease mucosa. Mucosal permeability changes were accompanied by mitochondrial swelling and a fall in epithelial ATP content, suggesting uncoupling of oxidative phosphorylation. CONCLUSIONS: The tight junctions in the non-inflamed distal ileum of Crohn's disease were more reactive to luminal stimuli, possibly mediated via disturbed cytoskeletal contractility. This could contribute to the development of mucosal neoinflammation in Crohn's disease.

Carrageenan-induced subacromial bursitis caused changes in the rat's rotator cuff.

This study was designed to investigate the histologic expression of the rat's supra- and infraspinatus tendons in carrageenan-induced subacromial bursitis. Thirty-two rats received subacromial injections with carrageenan (n = 28) or saline (n = 4). The tendons were analysed microscopically after staining with hematoxyline eosin, Van Giesons hematoxyline and immunofluorescent staining of fibronectin and fibrinogen. In the controls (saline x 10) and group A (carrageenan x 5) there were no changes in the tendons. In group B (carrageenan x 10) 3/8 rats showed macrophages between the collagen fibres and an increased staining of fibronectin. In group C (double dosis carrageenan) all rats had signs of fibrocartilaginous metaplasia in the supraspinatus tendon. In eight of these specimens even bony metaplasia was seen. The infraspinatus tendon showed fibrosis but no fibrocartilaginous metaplasia. The results showed that iatrogenic bursitis after carrageenan subacromial injections was associated with marked changes of the supraspinatus tendon.

Prevalence of gastroduodenitis and Helicobacter pylori infection in a general population sample: relations to symptomatology and life-style.

Some benign and malignant diseases develop on the background of chronic gastritis or duodenitis. The present study was performed in order to determine the magnitude of these background changes with relations to symptomatology and life style in the general population. Examinations were performed in 501 volunteers (age 35-85 years). Fifty percent had gastritis; this was associated with H. pylori in 87%. H. pylori-negative gastritis was associated with regular use of NSAIDs [odds ratio 3.8 (1.6-9.9)]. Duodenitis, observed in 32%, was associated with H. pylori infection [odds ratio 2.3 (1.3-4.6)], previous cholecystectomy [odds ratio 3.6 (1.1-16.1)], and regular use of NSAIDs [odds ratio 3.0 (1.4-7.1)]. Neither gastritis nor duodenitis was associated with smoking or alcohol consumption. The rate of digestive symptoms did not differ between subjects with and without uncomplicated gastritis or duodenitis. In conclusion, half of this adult population had gastritis strongly associated with H. pylori infection. Gastritis without H. pylori infection was frequently associated with regular NSAID intake. One third had duodenitis, which was associated with H. pylori infection as well as with regular use of NSAIDs and previous cholecystectomy. Digestive symptoms were not overrepresented in uncomplicated gastritis or duodenitis.

Effect of steroid injections on the rotator cuff: an experimental study in rats.

The aim of this study was to evaluate the effects of repeated steroid injections into the subacromial space. Thirty rats were injected either 3 or 5 times with triamcinolone in a dosage equivalent to that given to human beings or 3 or 5 times with saline into the subacromial space. One rat received no injection. The supraspinatus and infraspinatus tendons were evaluated macroscopically and microscopically. Two different staining methods were used on each sample including hematoxylin eosin and Miller's elastin/van Gieson's solution. After 5 steroid injections, we found focal inflammation, necrosis, and fragmentation of collagen bundles in the tendon in 4 of 7 rats. The tendons of the controls showed a normal structure (P < .05). There were no pathologic changes among the rats that were injected with triamcinolone 3 times. These results show that repeated subacromial injections of triamcinolone may cause damage to the rotator cuff of the rat. This finding may indicate cautious use of subacromial steroid injections in human beings.

Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD. METHODS: Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy. RESULTS: Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria. CONCLUSIONS: Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.

The clinical significance of slightly to moderately increased liver transaminase values in asymptomatic patients.

BACKGROUND: Our aim was to study liver disorders in asymptomatic patients with slightly to moderately increased liver transaminase values in a population living in an area with a low prevalence of viral and hereditary liver diseases. METHODS: One hundred and fifty consecutive patients with slightly to moderately increased liver transaminases for at least 6 months without symptoms or signs of liver disease were included. Median (range) was 0.75 microkat/l (0.24-2.9) for aspartate aminotransferase (ASAT) and 1.18 microkat/l (0.28-4.5) for alanine aminotransferase (ALAT). A percutaneous liver biopsy was performed, and blood was sampled for a detailed biochemical and serologic profile. RESULTS: Chronic viral hepatitis C was found in 15.3% of the patients, autoimmune hepatitis in 1.3%, primary biliary cirrhosis in 1.3%, and heterozygotic alpha-1-antitrypsin deficiency in 0.7%. Presumed alcoholic liver disease was diagnosed in 8%, and non-alcoholic steatohepatitis in 2%. Chronic hepatitis with no obvious etiology was diagnosed in 24%, of whom 39% had interface hepatitis (piecemeal activity). Seventy-one per cent of these 39% had measurable levels of autoantibodies, but IgG levels within normal limits prevented the 'clinical' diagnosis of autoimmune hepatitis. Liver steatosis was the diagnosis in 40%. Most were overweight and had increased serum triglyceride levels. However, in 13.3% the fatty infiltration was considered 'essential', as both body mass index (BMI) and triglyceride levels were normal. Other diagnoses were liver fibrosis with no obvious inflammatory activity (3.3%), cirrhosis of unknown etiology (0.7%), and for the remaining (3.3%) patients histopathologic findings were considered 'normal'. Cirrhosis was found in five biopsy specimens: hepatitis C (n = 2), autoimmune hepatitis (n = 1), primary biliary cirrhosis (n = 1), and cryptogenic cirrhosis (n = 1). No concomitant disease was of importance for the diagnosis and/or histopathologic findings. No obvious drug-related increased liver test results were found with any single drug. However, patients with chronic hepatitis of unknown etiology, especially with interface hepatitis, significantly more often than the rest of the population were receiving drug treatment. CONCLUSION: Most transaminitis patients had steatosis, and some had defined diseases including chronic hepatitis C. Chronic hepatitis of unknown etiology was found in a substantial proportion (24%) of a population living in an area with a low burden of hepatic viruses and genetic disorders.

Fibroblast movements during contraction of collagen lattices--a quantitative study using a new three-dimensional time-lapse technique with phase-contrast laser scanning microscopy.

In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction. Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16-26 "optical sections" 5 microm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices decreased by a mean of 6.8 microm, but remained constant in the anchored group. Only minor variations were found in the angle between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions during lattice contraction.

Expression of alpha-smooth muscle actin, TGF-beta 1 and TGF-beta type II receptor during connective tissue contraction.

Closure of rat mesenteric perforation is considered to occur by connective tissue contraction, a process that has been shown to be stimulated by transforming growth factor-beta 1. In the present study, we assessed the expression of alpha-smooth muscle actin during closure by quantitative-reverse transcription-polymerase chain reaction and in situ hybridization. The expression of transforming growth factor-beta 1 and transforming growth factor-beta type II receptor was also estimated in mesenteric membranes and free peritoneal cells after wounding. A larger expression of alpha-smooth muscle actin was seen around the wound edges compared to unwounded tissue. Both alpha-smooth muscle actin and transforming growth factor-beta type II receptor were expressed during Days 0, 3, 5, 7, and 10. The expression of alpha-smooth muscle actin on Day 5 was > 100 times higher than on Day 0. Transforming growth factor-beta 1 was expressed in both membranes and free peritoneal cells of unoperated control animals but down-regulated after wounding, a finding that has not been reported previously. It reappeared on Days 7 and 10 in free peritoneal cells but not in perforated membranes. The enhanced expression of alpha-smooth muscle actin and down-regulation of transforming growth factor-beta 1 expression after wounding appears to be important phenomena in tissue contraction and repair.

Alpha-smooth muscle actin expression in rat and mouse mesenteric wounds after transforming growth factor-beta1 treatment.

Rat mesenteric perforations heal by contraction within 5 to 7 days, whereas mouse mesenteric perforations seldom close within 3 weeks unless stimulated by transforming growth factor-beta1. In this article, we quantified the expression of alpha-smooth muscle actin by quantitative-reverse transcription-polymerase chain reaction and the orientation of actin filaments at the wound margin by Fourier transformation image analysis after treatment with transforming growth factor-beta1. The expression of transforming growth factor-beta1 and its type II receptor was also assessed. Actin filaments were shown to increase with time at the wound margin in both species and the expression of alpha-smooth muscle actin mRNA increased simultaneously. Transforming growth factor-beta1 enhanced the alpha-smooth muscle actin expression four to five times in rats and three to four times in mice on day 5, but the number of copies expressed per cell was 15-fold higher in rats than in mice. Transforming growth factor-beta1 was down-regulated after wounding in free peritoneal cells of rats, but maintained until day 5 in transforming growth factor-beta1-treated mice. The main finding of this study was that untreated, normal rats expressed substantially more alpha-smooth muscle actin than mice. After treatment with transforming growth factor-beta1, this expression increased similarly in both species. It can be hypothesized that normal closure of mesenteric perforations requires a minimum level of actin expression. This level is not reached in normal mice, but is exceeded after stimulation. Perforations in the rat always close, because the alpha-smooth muscle actin expression is always above this level.

Stimulation of protracted connective tissue repair in normal mice by transforming growth factor beta 1.

The repair and contraction during connective tissue repair of mesenteric perforations is prolonged in mice compared with rats. In the present study the stimulating effect of transforming growth factor beta 1 (TGF-beta 1) on different aspects of such repair of the mouse mesentery was assessed. The number of closed mesenteric perforations were counted on different days after operation and the free peritoneal cells were counted, the mitotic index was assessed, and actin distribution of fibroblasts around the perforation was studied with laser scanning confocal microscopy. TGF-beta 1 significantly increased the speed of closure and seemed to induce more actin in fibroblasts at the wound margin. It did not significantly influence the mitotic index, but fewer free peritoneal cells were obtained in mice treated with TGF-beta 1. We conclude that TGF-beta 1 is a potent stimulator of connective tissue repair and contraction in mice. The different methods of closure in rats and mice implicate different molecular responses in wounds and further studies on the stimulating effect of TGF-beta 1 may indicate basic fibroblastic cellular mechanisms that are active during contraction in connective tissue repair.

Actin fiber orientation in connective tissue contraction: a quantitative study with the perforated rat mesentery model.

Numerous fibroblasts with long cytoplasmic protrusions containing F-actin appear during closure by contraction of rat mesenteric perforations. These protrusions are predominantly arranged parallel to the wound margin. In the present study, the spatial and temporal organization of such protrusions was quantitated during normal and retarded healing in zinc-deficient animals. An orientation index, which gives the global orientation of the F-actin fibers, was calculated after Fourier transformation of images generated in a laser confocal microscope. Actin-rich fibroblasts began to accumulate at the wound margin on the first postoperative day, and the amount of actin gradually increased with time. The orientation index of the F-actin in unperforated mesenteric membranes was low, whereas the orientation index around perforations increased significantly (p < 0.001) with time and reached a maximum level on postoperative days 5 to 7. The orientation index was significantly lower (p < 0.02) in membranes with retarded healing in zinc-deficient animals than in controls. The findings show that image analysis can give valuable information about fibroblast organization during tissue repair and indicate that the spatial organization of the fibroblasts is an important factor in connective tissue contraction.

The perforated mesentery of the rat: a novel model for the study of genuine connective tissue contraction.

The rat and mouse mesenteries consist of an array of thin, mesenchymally derived, connective tissue membranes. Perforations of rat membranes heal by closure within a week and the perforated rat mesentery has earlier been shown to be a very suitable model of true connective tissue repair. In the present study, the closure of perforations in the rat was studied by light and transmission electron microscopy as well as confocal microscopy after actin staining with phallacidin in order to better understand the cellular mechanisms of healing in this model. The healing of different sized mesenteric perforations was also quantitatively assessed and compared in rats and mice. Closure occurred rapidly between Days 5 and 7 in rats, the velocity of healing being dependent on the size of the wounds. During closure, fibroblasts close to the wound margin ultrastructurally showed long slender cytoplasmic processes that contained actin filaments as shown by fluorescence confocal microscopy. In mice, larger perforations of mesenteries decreased in size during the 3-week observation period, but very seldom closed completely. In conclusion, the data gathered now and earlier indicate that a contraction phenomenon is of major importance in the closure of rat mesenteric perforations. Until now, good models for genuine connective tissue contraction have been lacking and it is suggested that the perforated rat mesentery may be used as such a model system in the future. The finding that mouse mesenteric perforations normally do not heal will make comparative studies intriguing, but it also indicates basic differences between rats and mice with respect to connective tissue repair mechanisms.

Mechanisms of TGF-beta action in connective tissue repair of rat mesenteric wounds.

We have recently reported that transforming growth factor-beta stimulates genuine connective tissue repair in the perforated rat mesentery and that this stimulation is not caused by increased macrophage chemotaxis. To further characterize the effect of transforming growth factor-beta(1) on the enhanced rate of wound closure, we performed a series of morphometric analyses with determination of mitotic index, fibroblast labeling index, cellular density, neovascularization, and scar tissue formation. Actin expression close to the wound margin was also evaluated morphologically. Fibroblast cell proliferation was not stimulated by transforming growth factor-beta(1) in either wounded or unwounded tissue. Transforming growth factor-beta(1) did, however, significantly increase the formation of healing tissue postoperative days 5 to 10 (p < 0.05) and angiogenesis was significantly stimulated by transforming growth factor-beta(1) postoperative days 7 and 10 (p < 0.005). The mean cellular density was significantly increased in unperforated, transforming growth factor-beta(1)-treated membranes from days 3 to 10, and increased expression of actin with time was observed close to the wound margin. Transforming growth factor-beta(1) was thus shown to be a potent stimulator of angiogenesis and healing tissue formation in connective tissue repair, but this stimulation mainly occurred after closure of perforations. The increased cellular density in the absence of stimulated proliferation and increased actin expression in wound cells indicate that contraction may be an important mechanism of connective tissue repair in the perforated rat mesentery.

Impaired function of postoperative macrophages from zinc-deficient rats decreases collagen contraction. Brief report.

Zinc deficiency impairs connective tissue contraction in the perforated rat mesentery model. Since the rat mesentery is almost avascular, free peritoneal macrophages are important for mesenteric repair. Impairment of contraction may thus be caused either by a direct effect of zinc deficiency on tissue cells or by hampered macrophage function. To further elucidate the role of macrophages in tissue contraction, we studied their effect on lattice contraction. A number of typical functions of macrophages in zinc deficiency were also investigated. Lattice contraction was significantly impaired by conditioned medium from zinc-deficient macrophages. Zinc deficiency did not influence peripheral blood leukocyte number, but postoperatively the number of peritoneal macrophages increased on days 7 and 10. A significant release of lysosomal enzymes from macrophages was recorded during phagocytosis, whilst no difference was observed between controls and zinc-deficient macrophages. Superoxide anion generation during phagocytosis was not significantly increased in zinc deficiency. Conditioned medium from zinc-deficient macrophages was shown to impair lattice contraction in vitro and the results are compatible with impaired macrophage function as a cause of decreased connective tissue contraction in vivo.

Impaired connective tissue repair in streptozotocin-induced diabetes shows ultrastructural signs of impaired contraction.

Streptozotocin-induced diabetes mellitus is known to impair connective tissue repair in the perforated rat mesentery. The aim of the present investigation was to study quantitatively by morphometrical techniques the influence of diabetes on some aspects of the cellular ultrastructure related to connective tissue contraction in such healing. The cellular volume density increased significantly with time, presumably as a consequence of disappearance of interstitial edema. No difference was found in the amount of healing tissue formed between controls and diabetics. The surface volume density of the plasma membrane was significantly higher in control animals on Days 1-10, indicating an increased number of cellular protrusions and spikes which relate to the motility of the cells. The volume density of contractile filaments did not differ between controls and diabetics. The results suggest a reduced surface density of plasma membrane in diabetic cells, a finding which is compatible with reduced wound contraction in diabetes.

Transforming growth factor-beta enhances connective tissue repair in perforated rat mesentery but not peritoneal macrophage chemotaxis.

The perforated rat mesentery model was used to study the effect of transforming growth factor-beta (TGF-beta) on connective tissue repair and influx of macrophages into the peritoneal cavity during such repair. Sprague-Dawley rats were laparotomized, and mesenteric wounds were made with a scalpel. A daily intraperitoneal injection of 0.5 microg TGF-beta was given for either 2 or 4 days. After 1 to 10 days, the animals received an intravenous injection of tritium-labeled thymidine before decapitation. Macrophages were collected by peritoneal washing, and the number of closed perforations was counted. Peritoneal cells were quantitated and a labeling index was determined by autoradiography. TGF-beta given for either 2 (p < 0.001) or 4 (p < 0.004) days accelerated closure of perforations on days 3 to 7 after injury. Laparotomy as such significantly increased leukocyte influx (p < 0.004), as well as macrophage-labeling index (p < 0.02). However, TGF-beta did not significantly influence either leukocyte influx or macrophage-labeling index. We concluded that TGF-beta significantly enhances connective tissue repair in this perforated rat mesentery model and that TGF-beta-induced stimulation of repair is not caused by an increased influx of macrophages into the peritoneal cavity.