Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.

TGF-beta1 functions as a negative regulator of T cell immune responses, signaling to target cells using the Smad family of proteins. We show here that Smad7, an inhibitor of TGF-beta1 signaling, is overexpressed in inflammatory bowel disease (IBD) mucosa and purified mucosal T cells. Both whole tissue and isolated cells exhibit defective signaling through this pathway, as measured by phospho-Smad3 immunoreactivity. Specific antisense oligonucleotides for Smad7 reduce Smad7 protein expression in cells isolated from patients with IBD, permitting the cells to respond to exogenous TGF-beta1. TGF-beta1 cannot inhibit proinflammatory cytokine production in isolated lamina propria mononuclear cells from patients with Crohn disease (CD), but inhibition of Smad7 restores TGF-beta1 signaling and enables TGF-beta1 to inhibit cytokine production. In inflamed mucosal tissue explants from patients with CD, inhibition of Smad7 also restores p-Smad3 and decreases proinflammatory cytokine production, an effect that is partially blocked by anti-TGF-beta1. These results show that Smad7 blockade of TGF-beta1 signaling helps maintain the chronic production of proinflammatory cytokines that drives the inflammatory process in IBD and that inhibition of Smad7 enables endogenous TGF-beta to downregulate this response.

Helicobacter pylori colonization density and gastric acid output in non-ulcer dyspepsia and duodenal ulcer disease.

BACKGROUND: The pattern of intragastric Helicobacter pylori colonization and its density may be determined by parietal cell function. H. pylori bacterial products can inhibit gastric acid secretion from the parietal cell. The aim of this investigation was to study the relationship between acid output and intragastric H. pylori distribution and colonization density in duodenal ulcer (DU) and non-ulcer subjects. The study included 14 patients with active DU, 10 with inactive DU and 10 non-ulcer dyspeptics. METHODS: Acid output studies in response to fasting and maximal pentagastrin stimulation, basal (BAO) and peak (PAO) acid outputs were calculated. A quantitative assessment of H. pylori colonization density in biopsies from five sites of the gastroduodenum in the active ulcer group, and from the antrum in inactive duodenal ulcer and non-ulcer groups. RESULTS: There were negative correlations between total gastroduodenal bacterial colonization density and, PAO (r - 0.87, p = 0.0025) and BAO (r - 0.635, p < 0.02) in the active ulcer group. There were negative correlations between antral H. pylori colonization density and PAO in the active duodenal ulcer (r - 0.7449, p < 0.01) and non-ulcer (r - 0.5837, p < 0.1) groups but not in the inactive duodenal ulcer group (r - 0.1869, p > 0.2). CONCLUSIONS: An equilibrium is reached between gastroduodenal H. pylori colonization density and gastric acid secretory capacity in active duodenal ulcer disease. It is hypothesized that thresholds of bacterial load and acid secretory capacity, in combination, are required for active ulceration in DU disease.

Increased gastric epithelial cell proliferation in Helicobacter pylori associated follicular gastritis.

AIMS: An increase in the proliferative state of the gastric epithelium has been attributed to infection with Helicobacter pylori. In order to obtain a more precise estimate of the magnitude of this change, the proliferative state of 17 cases of florid H pylori associated follicular gastritis was examined using the antibody MIB-1. METHODS: Comparable results were produced from control and gastritis cases by using a combination of two reproducible measures of the labelled cells. Dividing cells in the gastric mucosa are concentrated within a proliferating compartment, situated at the base of the crypts. This compartment was measured and expressed as a proportion of the total crypt length. The proportion of positively labelled cells within the compartment was also counted. RESULTS: The proliferation compartment in the gastritis cases occupied 45.6% of the gastric crypt compared with 15.4% in the control group. Of the cells in the proliferating compartment, 79.5% were positively labelled in the gastritis cases and 33.4% in the control group. CONCLUSIONS: The convoluted nature of the gastric crypt does not make it a forgiving experimental model. The use of long lengths of mucosa obtained from gastrectomy specimens permitted the production of consistent results, using a morphometric method. The greater than 100% difference in the proportion of proliferating cells between the two groups suggests that further investigation is warranted.

Germinal centre and B lymphoblast responses in the parathymic lymph nodes after localized peritoneal inflammation.

Inflammatory cells from a focus of peritoneal inflammation are known to migrate from the peritoneal cavity to the parathymic lymph nodes inducing a B-lymphocyte response in the medulla. The present study demonstrates that the medullary response is preceded by an expansion of the germinal centres with evidence of blast transformation. The number of cells in the lymphocytic corona is increased with a significant increase in the percentage of large cells containing immunoglobulin. This cellular expansion involves immunoglobulin M and immunoglobulin G containing cells.

Rare intrathoracic complications in acute pancreatitis.

Ascites and pleural effusions may complicate pancreatitis but pericarditis with pericardial effusion and tamponade is rare and necrosis of mediastinal fat has not been described before. All these complications occurred in the case reported here.

Changes in the medulla of the parathymic lymph nodes of the rat during acute gastro-intestinal inflammation.

The changes occurring in the medullary sinus and medullary cord of the parathymic lymph nodes were studied in rats after performing a small intestinal anastomosis. Polymorphonuclear leucocytes and eosinophils were phagocytosed by the macrophages of the medullary sinus. The sinus macrophages stained strongly for acid phosphatase and weakly for non-specific esterase. The medullary sinus macrophages were most numerous near the subcapsular sinus. After a small intestinal anastomosis the sinus macrophages increased in size and in number. The macrophages of the medullary cord differed from those of the medullary sinus both histochemically and ultrastructurally. The cord macrophages stained weakly for acid phosphatase, stained strongly for non-specific esterase and phagocytosed plasma cells, particularly in those specimens 2 and 5 days after the intestinal anastomosis. There was an initial significant decrease in IgA plasma cells at 2 days after the anastomosis but a subsequent increase in IgG and IgM plasma cells. The increase in IgM plasma cells preceded that of the other plasma cells.

Tumour cell trapping in rat mesenteric lymph nodes.

We describe a new experimental model of mesenteric lymph node metastasis in the rat, involving afferent mesenteric lymphatic inoculation of tumour cell suspensions via glass microcannulae. This model has been used to perform a series of experiments to investigate whether the rat mesenteric lymph node trapped tumour cells. Afferent mesenteric lymphatic inoculation of suspensions of transplantable sarcoma cells in inbred hooded Lister rats resulted in tumour growth in the inoculated lymph node in 100% of rats, with no tumour growth at any other site. The same procedure performed on rats which had previously undergone mesenteric lymphadenectomy resulted in growth of tumour in the lungs. Using 125Iododeoxyuridine (IDUR) labelled sarcoma cells we have shown that although radioactivity decreased significantly in the mesenteric lymph node up to 24 h following afferent lymphatic inoculation, there was no evidence that tumour cells reached thoracic duct lymph. We conclude that the rat mesenteric lymph node trapped sarcoma cells.

Transplantable colonic adenocarcinomas in rats.

The induction of colonic adenocarcinoma using two different regimens of dimethylhydrazine (DMH) in Fischer F344 rats is described. Rats receiving 20 mg/kg of DMH per week for 20 weeks developed primary tumors with metastases, whereas rats receiving the same weekly dose for 15 weeks developed primary tumors only. The most common route of metastases was transcelomic which often was associated with ascites. The epithelial origin of malignant ascites cells was confirmed by immunofluorescent staining with antidesmosomal antibodies and demonstration of desmosomes by electron microscopy. When transplanted into syngeneic rats, the cells of the malignant ascites resulted in the development of adenocarcinomatous metastases.

The gastro-duodenal epithelium in peptic ulceration.

Mucosa-related bacteria, intra-epithelial lymphocytes and intra-epithelial polymorphonuclear leucocytes have been studied in 35 patients with duodenal ulceration, 27 patients with gastric ulceration and eight control subjects with normal gastro-duodenal mucosa. Mucosa-related bacteria were found in approximately 80 per cent of peptic ulcer patients and rarely in controls. The bacteria were most numerous at the sites of active chronic gastritis. There was a positive correlation between the number of bacteria and the number of intra-epithelial polymorphonuclear leucocytes. There was no correlation between the peripheral blood white cell count and the number of intra-epithelial polymorphonuclear leucocytes. The number of intra-epithelial lymphocytes was increased in peptic ulceration.

Surface morphology of the gastroduodenal mucosa in duodenal ulceration.

Endoscopic biopsies from the duodenal cap and prepyloric areas of 25 patients have been examined with the scanning electron microscope. Eleven patients had duodenal ulceration. Bacteria are related only to the surface of gastric type epithelial cells whether these cells are located at areas of gastric metaplasia in the duodenal bulb or in the pre-pyloric region of the stomach. The bacteria are not associated with the surface of intestinal type epithelial cells. The bacteria are absent from the biopsies of those patients with a normal stomach and duodenum. Of those patients with duodenal ulceration, 73% have bacteria related to the epithelial surface. The bacteria are of two morphological types - a kidney shaped bacillus and an S-shaped bacillus.

Peritoneal cell responses to acute gastro-intestinal inflammation.

Cell changes in the peritoneal exudate induced by an acute localised inflammation due to a small intestinal anastomosis have been studied. The maximum response occurs on the second day. The neutrophil polymorphonuclear leucocyte output on the second is seven times greater in the peritoneal exudate than in the effluent lymph from the gastro-intestinal tract. The fate of the inflammatory cell response of the peritoneal exudate has been studied using thymidine-labelled cell transfer experiments. Some neutrophil polymorphonuclear leucocytes and eosinophils are phagocytosed by monocyte/macrophage cells in the peritoneal cavity. The majority of the neutrophil polymorphonuclear leucocytes and eosinophils migrate through diaphragmatic "stomata" to the parathymic lymph glands via the mediastinal lymphatics.

The influence of diabetes mellitus upon peripheral vascular disease.

The treatment of 332 consecutive patients referred to a peripheral vascular unit with rest pain, ulceration or gangrene of the lower limb has been studied. Ninety-seven (20 per cent) of the patients had diabetes mellitus. Although diabetes was related to adverse limb salvage and patient survival rates, this could be accounted for by the influence of initial presenting clinical features and treatment upon survival time. The influence of these factors upon survival time was still strong even when the data were stratified for diabetric status. Thus, an extensive amputation carries the worse prognosis, with a less extensive amputation intermediate between an extensive amputation and any other form of therapy. Patients with both rest pain and ulceration or gangrene have a poor prognosis when assessed by either limb salvage or mortality. The majority of the patients with less extensive amputations are diabetic. This is related to the high incidence of localized ulceration among those in the diabetic group. In diabetics, a less extensive amputation, if clinically indicated, is worthwhile because of the low incidence of a subsequent extensive amputation after a less extensive amputation and the better survival rate for those patients with less extensive amputations as compared with those with extensive amputations.

Characterization of nonlymphoid cells derived from rat peripheral lymph.

Mesenteric lymphadenectomy in rats is followed by union of peripheral and central lymphatics, allowing the collection of intestine-derived peripheral lymph cells via the thoracic duct for several days. These cells include a proportion of nonlymphoid cells (NLC) that show irregular and heterogeneous surface morphology including long pseudopodia and veils. They stain variably for nonspecific esterase and acid phosphatase and are ATPase-positive. Their nuclei are irregular and some contain cytoplasmic inclusions, some of which show peroxidase activity and/or contain DNA. NLC have a range of densitites generally lower than that of lymphocytes. Freshly collected NLC express the leukocyte-common antigen (defined by monoclonal antibody MRC Ox 1) and Ia antigens (I-A and I-E subregion products defined by monoclonal antibodies) but they show a relative lack of other surface markers normally found on rat B or T lymphocytes (W3/13, W3/25, MRC Ox 12 (sIg), MRC Ox 19) or rat macrophages (FcR, C'R, mannose R, W3/25). In general NLC are only weakly adherent to glass or plastic. Although a subpopulation of NLC appear to have had a phagocytic past, freshly collected NLC fail to phagocytose a variety of test particles in vitro. NLC also appear incapable of pinocytosis in vitro. This heterogeneity may represent distinct subpopulations of NLC or different stages in the development of a single cell lineage. Direct cannulation of mesenteric lacteals shows that the majority of NLC are derived from the small intestine and their precursors appear to be present both in lamina propria and Peyer's patches. Kinetic studies, following irradiation or intravenous tritiated thymidine, show that the majority of NLC turn over rapidly in the intestine with a modal time of 3-5 d. Studies with bone marrow chimeras show that they are derived from a rapidly dividing precursor present in normal bone marrow. NLC occur at very low frequencies in normal thoracic duct lymph at all times following cannulation. The evidence presented suggests that NLC closely resemble mouse lymphoid dendritic cells. This conclusion is supported by evidence already obtained showing that NLC are potent stimulators of the semi-allogeneic rat primary mixed leukocyte reaction. In addition to the ceils resembling dendritic cells rare monocytoid cells are found in thoracic duct lymph of lymphadenectomized specific pathogen-free rats. The proportion of these cells increases greatly when the animals are conventionally housed. It seems probable that the physiological function of NLC is to act as accessory cells in the lymph nodes to which they normally drain. Methods for enriching NLC and thus facilitating analysis of their functions are discussed.

The effect of intravenous Corynebacterium parvum on gut associated mononuclear phagocytes in normal and tumour bearing rats.

Corynebacterium Parvum, which has been used in the treatment of human colorectal cancer, probably exerts its action through cells of the mononuclear phagocyte system (MPS). In this study the effect of systemically administered C. parvum has been measured on gut associated MPS cells in normal and colorectal cancer bearing rats. MPS cells are not normally found in samples of lymph obtained after cannulation of the thoracic duct (TDC). However, after total extirpation of the mesenteric lymph nodes, TDC yields samples in which up to 5% of the total cell population appear to be MPS cells. This procedure has been carried out in adult Wistar rats enabling an in vivo study to be made on the effect of C. parvum treatment on the effluent gut cells. Measurements have been made both of the number of cells found in thoracic duct lymph and of their capacity to phagocytose sensitized sheep red blood cells. These measurements were repeated in a mesenteric lymphadenectomized group of rats which had also undergone induction of colonic cancer using dimethylhydrazine. C. parvum treatment did not effect total cell, or phagocyte numbers in thoracic duct lymph (TDL). However rats with colonic cancers showed a marked reduction in the numbers of phagocytic cells in TDL irrespective of C. parvum treatment.

Observations on the immunocytes and macrophages in megacolon.

An immunoperoxidase staining method was used to study specific immunoglobulin-containing cells in the intestinal mucosa of children who presented with features suggestive of Hirschsprung's disease. No evidence was found to substantiate the hypothesis of an immunologic component in the etiology of Hirschsprung's disease. The megacolon in this condition had an increased proportion of IgG-containing cells and a reduced proportion of IgA-containing cells when compared with normal and unobstructed colon; this may be a reflection of an abnormal mucosal immune defense leading to susceptibility to the enterocolitis found in Hirschsprung's disease. Numerous lipofuscin-bearing macrophages were seen in the colonic mucosa of children with anal stenosis who were given anthraquinone laxatives for varying periods. The possibility that this represents an early stage of melanosis coli, as well as the likelihood of anthraquinones contributing to the pathogenesis of acquired megacolon, are considered.

Studies of the immunoglobulin-producing cells of the human intestine: the defunctioned bowel.

An indirect immunoperoxidase method was used to visualise immunoglobulin-containing cells in the large intestinal mucosa of 10 children who had defunctioning colostomies. Intestine deprived of its usual exposure to intraluminal antigens contained less immunocytes per unit area than intestinal mucosa subjected to normal stimulation by dietary and microbial antigens. These findings substantiate in man the conclusion based on observations made on animals that continued mucosal exposure to antigenic stimulation is necessary for the existence of an adequate population of intestinal immunocytes.

Study of acute localised inflammation of the gastrointestinal tract: the effluent lymph.

The effect of localised acute inflammation, produced by a small intestinal anastomosis on the effluent lymph of the gastrointestinal tract and on the efferent lymph of the mesenteric lymph glands has been studied in rats. There is a progressive increase in the output of lymph from the gastrointestinal tract in rats with an intact anastomosis, but a decreased output in animals with a disrupted anastomosis causing either generalised peritonitis or a localised para-anastomotic abscess. The total white cell output is increased on the second day after constructing an intact intestinal anastomosis and this increase is principally due to neutrophil polymorphonuclear leucocytes. The neutrophil polymorphonuclear leucocyte response is prolonged, but has returned to normal values at four weeks. Although the output of cells of the mononuclear phagocytic series which are esterase positive is increased it is not statistically significant. An intact anastomosis does not produce any alteration in the lymphocyte output. The neutrophil polymorphonuclear leucocyte response to an intestinal anastomosis is decreased by a factor of two and the non-lymphocytic non-specific esterase positive cell response is decreased by a factor of six by the mesenteric lymph glands which may be functioning in a 'filtering' capacity dealing with agents originating at the anastomosis and noxious to the body,