Regulation of matrix metalloproteinases in a model of colonic wound healing in a rabbit.

PURPOSE: Matrix metalloproteinases occur in the colon at an anastomosis but not in the normal colon. Matrix metalloproteinase synthesis can be regulated by cytokines, for example interleukin-1 beta and growth factors, such as transforming growth factor beta and basic fibroblast growth factor. The aim of this study was to investigate the regulation of matrix metalloproteinases at an anastomosis by identifying the cell types that synthesize matrix metalloproteinases, examining factors that might regulate their synthesis, determining whether they occur in an active form, and assessing the effect of suture type on these parameters. METHODS: An anastomosis was formed in the distal colon of rabbits using either polyglactin or polydioxanone and the animals were killed six hours or seven days later. The distribution of matrix metalloproteinases and cytokines and the cell types were assessed by immunohistochemistry. Matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were detected also by zymography. RESULTS: Immunohistochemistry showed that matrix metalloproteinases were restricted to the suture line. Although zymography demonstrated that matrix metalloproteinase-2 was present mainly in an active form, matrix metalloproteinase-9 and matrix metalloproteinase-3 were present in the pro-form. The active form of matrix metalloproteinase-3 occurred more often in the polydioxanone-sutured rabbits. With the exception of matrix metalloproteinase-9, the matrix metalloproteinases were synthesized by fibroblasts. Interleukin-1 beta and transforming growth factor beta were more widespread than in the normal colon and were localized adjacent to the matrix metalloproteinases. Basic fibroblast growth factor was also more widespread postoperatively but occurred deeper in the anastomosis than the matrix metalloproteinases. CONCLUSIONS: This study has shown that interleukin-1 beta and transforming growth factor beta may regulate the synthesis of the matrix metalloproteinases by fibroblasts and that minor differences that occur in the matrix metalloproteinase profile are dependent on the suture type.

Endothelin receptor expression in colorectal cancer.

The distribution of endothelin-A- and B- (ET(A), ET(B)) receptor subtypes was compared in colorectal cancer to that in normal colon and their expression in the colorectal cancer cell lines LIM1215. HT29, SKCO1, SKCO17 and LoVo was determined, using gross and high resolution autoradiography and quantified by densitometry. ET(A) and ET(B) binding sites were expressed by all the cell lines. There was significantly (p = 0.008) higher expression of ET(A)-receptors by cancers (205.95 dpm x 1000/mm2) compared normal colon (129.19 dpm x 1000/mm2). However, for ET(B)-receptors, this was reversed, with significantly (p = 0.008) higher expression of ET(B) binding in normal colon (207.00 dpm x 1000/mm2) than in tumours (122.35 dpm x 1000/mm2).

Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ET(A) antagonists.

BACKGROUND: The vasoactive peptide endothelin 1 (ET-1) acts via two receptors, endothelin receptors A (ET(A)) and B (ET(B)). ET-1 is overexpressed by human cancers in vivo and in vitro and may be mitogenic for cancer cells. METHOD: To elucidate if ET-1 is a growth regulator the following were investigated in human colorectal cancer cell lines (LIM1215 and HT29): ET-1 production by ELISA; ET receptor expression using radioligand autoradiographic techniques; and responsiveness to ET-1, and to ET(A) and ET(B) antagonism by growth measurements. RESULTS: ET-1 was produced by LIM1215 and HT29 cells (21.3 and 41.7 fmol/ml/10(6) cells (24 hours); 22.6 and 71.7 fmol/ml/10(6) cells (48 hours), respectively). ET(A) and ET(B) receptors were expressed by both cell lines. Addition of ET-1 resulted in a dose dependent increase in cell numbers which was significant at 10(-8)-10(-9) M for LIM1215, with the greatest increase at 10(-8) M (32.7% and 28.4% increase above controls at 48 hours and 72 hours; p<0.05) and at 10(-8)-10(-9) M for HT29, with the greatest increase at 10(-9) M (13.4% and 15.7% increase above controls at 48 hours and 72 hours; p<0.05). ET(A) antagonists BQ123 and BQ610, but not the ET(B) antagonist BQ788, inhibited ET-1 induced proliferation of both LIM1215 and HT29 (p<0.05). CONCLUSION: ET-1 can stimulate the proliferation of colorectal cancer cell lines via the ET(A), but not the ET(B), receptor.

Counselling on breastfeeding: assessing knowledge and skills.

Reported are the results of a randomized controlled trial to assess the effectiveness of the WHO/UNICEF 40-hour course "Breastfeeding counselling: a training course". The course was conducted in a maternity hospital which provides care to a low-income population in a metropolitan area in São Paulo, Brazil. Health workers from 60 health units were randomly assigned to be either participants (20) or controls (40), and their breastfeeding knowledge and skills were assessed before and immediately after the course, as well as 3 months later. Immediately after the course the participants' knowledge of breastfeeding had increased significantly compared to controls. Both their clinical and counselling skills also improved significantly. When assessed 3 months later, the scores remained high with only a small decrease. The implementation of the course was also evaluated. The methods used were participatory observation, key interviews and focus group discussion. In the 33 sessions of the course, the average score was 8.43 out of 10. Scores were highest for content and methodology of the theory sessions, and lowest for "use of time", "clinical management of lactation", and "discussion of clinical practice". "Breastfeeding counselling: a training course" therefore effectively increases health workers' knowledge and their clinical and counselling skills for the support of breastfeeding. The course can be conducted adequately using the material and methodology proposed, but could be more satisfactory if the time allocated to exercises and clinical practice sessions were increased.

PIP: This document presents a report which assesses the effectiveness of the WHO/UNICEF 40-hour course "Breastfeeding counseling: a training course" (BFC). The course was conducted in a maternity hospital which provides services to a low-income population in Sao Paulo, Brazil. The randomized controlled trial was composed of 60 health professionals divided into an "exposed" group (20) and a control group (40). The participants' breastfeeding knowledge and skills were assessed before, immediately after, and 3 months after the course. Results showed that the participants' knowledge of breastfeeding together with their clinical and counseling skills had markedly improved by the period immediately after the course. Three months after the course, their knowledge skills remained high with only a slight decrease. Participatory observation, key interviews and focus group discussions were used in evaluating the course implementation. The content and methodology of the theory sessions received the highest scores whereas "use of time", "clinical management of lactation", and "discussion of clinical practice" got the lowest scores. In general, BFC was effective in increasing the health workers' clinical and counseling skills for the support of breastfeeding. The course, however, does need to be improved with regard to the time allocated for exercises and clinical practice sessions.

Effect of colonic obstruction on the distribution of matrix metalloproteinases during anastomotic healing.

BACKGROUND: Anastomotic dehiscence is common after surgery for colonic obstruction. The strength of an anastomosis is dependent on collagen, which is degraded by matrix metalloproteinases (MMPs). The aim of this study was to determine the distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP) 1 in an experimental model of colonic obstruction, with and without resection and anastomosis. METHODS: The distal colon of rabbits was obstructed with a Silastic ring for 24 h and then either the ring was removed or the obstructed segment was resected and an anastomosis formed. Rabbits were killed immediately or at intervals for up to 7 days after operation. The distribution of the MMPs and TIMP-1 was examined by indirect immunofluorescence. RESULTS: MMPs and TIMP-1 were present throughout the descending colon for 24 h in both groups. They persisted to the third day in rabbits with an anastomosis but by day 7 were restricted to the suture line. Their presence correlated with microscopic damage. CONCLUSION: The extensive distribution of the MMPs suggests that these enzymes contribute to anastomotic dehiscence, but only in the immediate postoperative period.

Role of matrix metalloproteinases in healing of colonic anastomosis.

PURPOSE: The aim of this study was to compare the distribution of the matrix metalloproteinases (MMPs) during anastomotic healing in a normal colon with that in an ischemic colon in a rabbit. This family of enzymes degrades all components of connective tissue and has been implicated as a cause of anastomotic dehiscence. METHODS: A left-sided anastomosis was formed in the distal colon of one group of rabbits, and in the other group, 9 cm of distal colon was made ischemic before resection and anastomosis 12 hours later. Tissues from the anastomosis and sites around the colon were removed at 12 hours, 1 day, and 3 days after anastomosis and, also, at 7 days in the normal group. Distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP), was localized by indirect immunofluorescence. RESULTS: In rabbits having only an anastomosis, the MMPs and TIMP-1 were, at all times, seen solely in the anastomotic segment and were strictly confined to the immediate vicinity of the suture line. While in rabbits with an ischemic colon before anastomosis, the MMPs initially extended several centimeters proximally and distally from the suture line. By the third day, however, there were only minor differences between the two models. CONCLUSION: Distribution of the MMPs and TIMP-1 in normal healing is consistent with a role in the remodeling of colonic anastomosis, but when healing of the colon is compromised, these enzymes are more widespread and may contribute to anastomotic dehiscence.

Effect of methylprednisolone on the ulceration, matrix metalloproteinase distribution and eicosanoid production in a model of colitis in the rabbit.

This study has examined the response of a rabbit model of inflammatory bowel disease to methylprednisolone. Colitis was induced in the colon of rabbits with 40 mg trinitrobenzenesulphonic acid in 25% ethanol (TNBS). The effect of methylprednisolone (0.5 mg/kg/day) on the development of colitis was determined at one week, by examining the colon's macroscopic and microscopic appearance, the distribution of matrix metalloproteinases (MMPs) and by measuring eicosanoid production. Although there was no difference in the area of ulcerated colonic tissue in the treated and untreated TNBS animals, the increase in polymorphonuclear leucocytes was significantly reduced in TNBS rabbits given methylprednisolone. The only difference in the distribution of MMPs was a reduction in the number of polymorphonuclear leucocytes containing gelatinase B. The release of immunoreactive PGE2 and LTB4, but not 6-keto PGF1 alpha, was increased in the TNBS animals and was unchanged by methylprednisolone. These results show that methylprednisolone does not modify the injury produced by TNBS in this model despite reducing the infiltration of polymorphonuclear leucocytes. Hence it suggests that these cells do not contribute to the injury observed, are not the source of the eicosanoids and that gelatinase B is not required in the healing process in this model.

The characterization of a rabbit model of inflammatory bowel disease.

The absence of a simple, clinically relevant, animal model of inflammatory bowel disease (IBD) hampers research into this disease. In this study, colitis was induced in rabbits by intracolonic installation of 2,4,6-trinitrobenzene sulphonic acid (TNB) in 25% ethanol. Rabbits were killed from zero hours to 6 weeks and their colons examined. Rabbits were examined by endoscopy at weekly intervals. A single dose of TNB in ethanol produced dose dependent inflammation and ulceration, which at its optimum (40 mg) resulted in cobblestoning, strictures, and bowel wall thickening. The damage score at endoscopy was consistent with the score on macroscopic examination of the colon. Histopathological features of inflammation and ulceration observed in all animals that received 40 mg TNB included crypt abscesses, ulceration, crypt architectural distortion and, occasionally, granulomas and pseudopolyps. These changes, which are similar to those observed in IBD, persisted for 6 weeks. No lasting abnormalities were observed in control animals treated with TNB in saline, with ethanol alone, or with saline only. Histopathological similarity and the prolonged duration of inflammation, compared to other models, make this a suitable model for investigating inflammation in the colon. Furthermore, the model is accessible to endoscopy which adds to its value in experimental studies.

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Training health care workers to counsel breastfeeding mothers.

PIP: WHO and UNICEF have designed a 40-hour counseling course for maternal-child health workers that aims to impart the skills needed to assist mothers to breast feed their infant. Course participants receive training in the following communication and counseling techniques: accepting what a mother feels as valid, recognizing and praising things a mother is doing right, giving practical guidance, using simple language, making suggestions rather than commands, and limiting the information provided so as not to overwhelm the mother. In addition, health workers are trained on the attachment and positioning techniques that promote successful breast feeding. Such techniques include having the infant's chin touch the breast, more areola exposed above than below the infant's mouth, close contact with the mother's body, and arrangement of the baby's head and body in a straight line. Common concerns, such as mothers' fears that they are not producing enough milk, sore nipples, and breast feeding practices when an infant is sick, are addressed. At the conclusion of the training, health workers apply the skills they have learned in maternity wards and maternal-child health clinics.^ieng

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumor cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Detection of free malignant cells in the peritoneal cavity before and after resection of colorectal cancer.

PURPOSE: This study was designed to select the best monoclonal antibody to stain malignant cells in peritoneal wash fluid, and to investigate the incidence of free malignant cells in preresection and postresection colorectal cancer peritoneal washings using a combination of conventional cytology and immunocytochemistry. METHODS: Peritoneal washings were taken from 35 consecutive patients undergoing colorectal cancer resection. RESULTS: Malignant cells were isolated on a density gradient and identified by conventional cytology and an indirect immunoperoxidase stain. Malignant cells were identified in peritoneal washings from 15 patients (preresection only n = 3, postresection only n = 4, both n = 8). The origin of free malignant peritoneal cells in 11 preresection-positive washings must be the serosa. The origin of these cells in the four postresection-positive patients is uncertain: serosal and luminal spillage were considered unlikely and no circulating cells were found in the mesenteric vessels near the tumor. CONCLUSION: Tumor cells may have leaked out from lymphatics cut during the dissection.

Loss of cell-cell and cell-matrix adhesion molecules in colorectal cancer.

Adhesion molecules are thought to play a vital role in the induction and maintenance of tissue differentiation and their loss or down-regulation has been implicated in the neoplastic process. Recent studies have shown that the morphoregulatory activities are a consequence of interactive processes between several cell adhesion molecules rather than the function of a single molecule. Therefore, we have investigated a panel of adhesion molecules including members of the integrin, cadherin and immunoglobin superfamily in colorectal cancer. Twenty-eight consecutive colorectal adenocarcinomas were stained using an avidin-biotin indirect immunoperoxidase technique. Our results showed a consistent loss of the alpha 2 and beta 1 integrin subunits (21/28 = 75% and 22/28 = 78.6% respectively) and a decrease in expression of E-cadherin in 5/5 poorly differentiated adenocarcinomas. Carcinoembryonic antigen expression was preserved but with basolateral accentuation seen in tumours. There was no statistical correlation with Dukes' stage. These results provide further evidence that in colorectal cancer there is a widespread deregulated expression of cell-cell and cell-matrix adhesion molecules. Changes in the expression and function of adhesion molecules which regulate growth and differentiation may play a role in the behaviour of colorectal cancer.

Nutrition. Milk the best way to feed.

PIP: Some problems with breast feeding lead mothers to supplement breast feeding with foods or fluids or to stop breast feeding before the end of the first 4-6 months of life. The leading reason mothers supplement early is that they believe they cannot produce enough breast milk. Health workers must determine whether or not a baby is receiving enough breast milk. If a breastfed baby receiving other fluids voids his/her bladder at least 6 times a day, the baby is receiving enough breast milk. A healthy baby should gain at least 500 g/month during the first 6 months of life. If a baby does not gain 500 g/month, he/she is either ill or is not receiving enough breast milk. If the baby's weight gain is appropriate, the health worker should encourage the mother to keep breast feeding. If the baby is not receiving enough breast milk, the health worker must determine if the baby is not suckling effectively or is not suckling long enough or if the mother is breast feeding less than 5-6 times a day and giving supplements. When suckling, the baby should take into his/her mouth a part of the breast tissue and the nipple. If the mother is not breast feeding often enough, the health worker needs to help the mother find ways to breast feed more often. If a mother works away from home and cannot take her baby to work, she can express milk so it can be given to the baby later. The health worker should inform the mother that she should let the baby breast feed until it stops suckling. She should not overwrap the baby during breast feeding since an overly warm baby falls asleep. If the baby falls asleep, the mother should rub his/her cheek to rouse the baby to continue breast feeding.^ieng

Detection and enumeration of circulating tumour cells in colorectal cancer.

Immunocytochemistry was used in parallel with conventional cytology to detect circulating malignant epithelial cells in 42 patients undergoing resection for colorectal cancer. Preoperative peripheral and peroperative mesenteric venous blood samples were taken. Tumour cells were isolated on a density gradient and cytospins prepared. Slides were stained by conventional cytology (May-Grünwald-Giemsa) and by an indirect immunoperoxidase technique with the anticytokeratin antibody KG8.13. Using conventional cytology, definite morphological evidence of malignancy was observed in three patients and suspicious features in a further seven. Immunocytochemistry confirmed these findings in all three of the malignant but in only one of the suspicious cases. Counts of immunostained cytospins showed the concentration of tumour cells in blood samples from these four patients to be in the range 0-954 cells/ml. This study supports the use of immunological markers to detect and enumerate malignant cells. This method provides a powerful tool to investigate one aspect of the metastatic process.

The chemoattractant activity of the vitreous to human scleral fibroblasts following retinal detachment and proliferative vitreoretinopathy.

The results are presented of a migration assay of scleral fibroblasts to 25% diluted vitreous samples from 27 patients with complex retinal detachments divided into three groups: group 1, no proliferative vitreoretinopathy (PVR); group 2, early PVR; and group 3, advanced PVR. There was a statistically significantly greater migration with vitreous samples from group 3 than with group 1 (p less than 0.0001) but no significant correlation for migration with the age of patients, duration of retinal detachment, or number of retinal procedures undertaken. Analysis of vitreous by gel electrophoresis showed that cellular migration was proportional to the number of peptide bands. Vitreous fibronectin levels were measured and there was a positive correlation between fibronectin in the vitreous samples and the migration of fibroblasts to the vitreous (r = 0.68, p less than 0.004).

Direct measurement of collagenase in colonic anastomosis.

Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radiochemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50-70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P less than 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P less than 0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.

The need for action.

Nutrition workers speculated about why poor mothers should decide to bottle feed, when their community in the past had apparently always breastfed effortlessly and successfully. Surveys cross-tabulated feeding methods against socioeconomic variables, but it was difficult to make use of the results. Few thought that there was anything we needed to learn about HOW to make breastfeeding succeed. There was also a tacit assumption that if you had the resources--for example if you were yourself an employed health worker--you could bottle feed safely, so there was no need to worry.