Differential expression of enteric neuroimmune-network in invasive and acute watery diarrhoea.

We aimed to evaluate the changes of nerve morphology and distribution of neurotransmitters and neuropeptides in the rectum of Shigella flexneri-infected patients and in the duodenum of Vibrio cholerae O1-infected patients. Nerve morphology was observed by transmission electron microscopy. Immunoreactivity of nerve growth factor (NGF), neurotransmitters and neuropeptides in tissues were studied by immunohistochemistry. Ultrastructural analysis of intestinal biopsy revealed persisting axons degeneration throughout the study period in all patients. Regeneration was already evident at the acute stage with marked increase at late convalescence. Both acute shigellosis and cholera were accompanied by increased expression of NGF and histamine and decreased expression of serotonin that was restored at convalescence. Immunoreactivity of vasoactive intestinal peptide (VIP) was increased during acute cholera, whereas in shigellosis VIP- and substance P-immunoreactive nerves appeared at early convalescence. Both shigellosis and cholera induced long-lasting degeneration of enteric neuronal axons, despite the presence of ongoing proliferation and regeneration processes. Neurotransmitters and neuropeptides may play differential roles in invasive and watery diarrhoea.

Biocompatibility of poly-DL-lactic acid (PDLLA) for lung tissue engineering.

This study explores the possibility of growing lung cells on poly-DL-lactic acid (PDLLA) scaffolds, with a view to in future engineer pulmonary tissue for human implantation. As a first step in this process, the ability of PDLLA to maintain the growth of lung epithelium is tested using a robust cell line. Poly-DL-lactic acid has been investigated in two forms, as planar discs and as 3-D foams, and it has been demonstrated that PDLLA is not only nontoxic to pneumocytes but it also actively supports their growth. The initial findings suggest that the material is an appropriate matrix for engineering of distal lung tissue.

Repopulation of human pulmonary epithelium by bone marrow cells: a potential means to promote repair.

After lung injury and damage to the alveolar epithelium, the underlying basement membranes become exposed. Proliferation of type II pneumocytes and their differentiation to the type I phenotype have been considered to be the mechanism by which repopulation of the alveolar epithelium occurs. A growing body of evidence has shown that tissues can be repaired by cells acquired via the circulation. For the lung, bone marrow stem cells have been shown in mice to regenerate epithelium as well as give rise to the expected mesodermal derivatives. We hypothesized that extrapulmonary cells, including those from the bone marrow, can contribute to the reepithelialization of human alveoli. To investigate this, we examined samples of peripheral lung from patients who had undergone cross-gender transplantation of lung or bone marrow. Thus, archival blocks of peripheral lung were analyzed from male patients (surgical samples, n = 8) who had received a lung transplant from a female donor and female patients (postmortem samples, n = 3) who had male bone marrow transplants. In both cases, male cells were identified in the female lungs by Y chromosome in situ hybridization. Male cells could be identified in the alveolar epithelium where, in the better preserved, transplanted lungs, it was possible to show that some had differentiated to type II pneumocytes. In addition, Y chromosomes were found to be widespread in cells of mesenchymal lineage, including macrophages and endothelial cells. Concomitant visualization of Y and X chromosomes, using fluorescence immunolabeling, yielded no evidence of cellular fusion, although the poor quality of the autopsy samples studied meant that the possibility could not be excluded. These observations suggest that, as occurs in rodents, the epithelium of the adult human lung has the capacity to renew itself, using cells recruited from extrapulmonary sources, including the bone marrow. This finding could provide new therapeutic opportunities for a range of pulmonary diseases by providing means to repair the lung and a novel route for gene therapy.

Coculture of embryonic stem cells with pulmonary mesenchyme: a microenvironment that promotes differentiation of pulmonary epithelium.

Coculture of stem/progenitor cells with mature cells or tissues can drive their differentiation toward required lineages. Thus, we hypothesized that coculture of murine embryonic stem (ES) cells with embryonic mesenchyme from distal lung promotes the differentiation of pneumocytes. Murine ES cells were differentiated to embryoid bodies (EBs) and cultured for 5 or 12 days with pulmonary mesenchyme from embryonic day 11.5 or 13.5 murine embryos, in direct contact or separated by a membrane. Controls included EBs cultured alone or with embryonic gut mesenchyme. Histology revealed epithelium-lined channels in directly cocultured EBs, whereas EBs grown alone showed little structural organization. The lining cells expressed cytokeratin and thyroid transcription factor 1, an early developmental marker in pulmonary epithelium. Differentiation of type II pneumocytes specifically was demonstrated by the presence of surfactant protein C (SP-C) in some of the epithelial cells. None of these markers was seen in EBs cultured alone or with embryonic gut mesenchyme. Indirect coculture of EBs with lung mesenchyme resulted in a 14-fold increase in SP-C gene expression. Thus, provision of an appropriate microenvironment, in the form of pulmonary mesenchyme, appears to promote the differentiation of ES cells toward lung epithelium. Our findings may have applications in regenerative medicine strategies and the engineering of lung tissue.

Pantoprazole in severe acid-peptic disease: the effectiveness and safety of 5 years' continuous treatment.

OBJECTIVE: This is our final report on the clinical effectiveness and safety of long-term pantoprazole in patients with severe peptic ulcer or reflux disease during continuous treatment for up to 5 years. METHODS: Patients (n= 150) with peptic ulcer or reflux erosive oesophagitis running an aggressive course or with complications, and refractory to H2-receptor antagonists, were entered into this 5-year programme. Assessment was by serial endoscopy, clinical examination, serum gastrin estimation, gastric mucosal histology and mucosal endocrine cell quantification. RESULTS: Healing results were presented earlier. The estimated rates of remission on maintenance treatment with pantoprazole (n = 115) were 82% at 1 year, 75% at 2 years, 72% at 3 years, 70% at 4 years and 68% at 5 years. Helicobacter pylori infection appeared not to influence the outcome in reflux patients, with roughly two-thirds continuing in remission irrespective of infection. Only four patients had adverse events considered to be definitely related to pantoprazole. Median gastrin levels rose by 1.5-2-fold and were higher in those with H. pylori infection; 13 patients had levels >500 ng/L on at least one occasion, but these high levels were not sustained. Histological changes were more marked in patients infected with H. pylori: chronic gastritis decreased in the antrum and increased in the corpus, which also showed atrophic changes. The total number of endocrine cells in the antrum showed little variation over 60 months but fell by around one-third in the corpus. CONCLUSION: Long-term treatment with pantoprazole is effective and safe.

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

A histological study of the hph-1 mouse mutant: an animal model of phenylketonuria and infantile hypertrophic pyloric stenosis.

AIM: To quantify the chronological sequence of changes in the morphology and immunoreactivity for neurotransmitters in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria. METHOD: Thirty specimens of pylorus from hph-1 mice and age/sex matched controls (age range: 10-180 days) were examined using conventional histology and immunohistochemistry for a variety of antigens: protein gene product 9.5, a pan neuronal marker; vasoactive intestinal polypeptide; nitric oxide synthase two antigens coalesced to the same inhibitory neurons in humans; substance P, a potent excitatory neurotransmitter; and calcitonin gene related peptide, a neurotransmitter implicated in the somatic afferent innervation of the stomach. The changes in the morphology of the muscle layers were quantified and statistically analysed for each age group (10, 20, 40, 90 and 180 days). RESULTS: Between 10 and 90 days of age, all muscle layers of the hph-1 mice were hypertrophied, for example, 10 days, hph-1 longitudinal muscle mean diameter = 3.4, control = 1.8; hph-1 circular muscle width = 11.5, control = 4.7. The hph-1 mice were significantly smaller during this period (40 days, hph-1 weight = 10 g, control = 25 g). There was no change in the pattern of expression of the antigens examined within the hph-1 mice compared with the controls. CONCLUSION: Hph-1 mice develop a transient smooth muscle hypertrophy of the pylorus attended by gastric distension and failure to gain weight. These changes resolve as the pyloric muscle hypertrophy resolves.

Embryonic stem cells.

Embryonic stem cells have huge potential in the field of tissue engineering and regenerative medicine as they hold the capacity to produce every type of cell and tissue in the body. In theory, the treatment of human disease could be revolutionized by the ability to generate any cell, tissue, or even organ, 'on demand' in the laboratory. This work reviews the history of murine and human ES cell lines, including practical and ethical aspects of ES cell isolation from pre-implantation embryos, maintenance of undifferentiated ES cell lines in the cell culture environment, and differentiation of ES cells in vitro and in vivo into mature somatic cell types. Finally, we discuss advances towards the clinical application of ES cell technology, and some of the obstacles which must be overcome before large scale clinical trials can be considered.

Pulmonary epithelial stem cells.

Classically, the stem/progenitor cells of the pulmonary epithelium are considered to be the basal and mucous cells of the proximal airways, Clara cells in the bronchioles and type II pneumocytes in the alveoli. Recent data suggest that there is a variant of Clara cells, lying in pulmonary neuroendocrine bodies, that meets several stem cell criteria and that type II pneumocytes exist in at least two populations, one of which is more resistant to injury. However, a complete revision of our understanding of pulmonary stem cell biology is underway as a result of the discovery of pulmonary epithelium derived from blood-borne cells. In addition, the existence in the lung of a 'universal' pluripotent cell has long been speculated upon and now some initial evidence has emerged with the identification of a spore-like cell that can differentiate in vitro to bronchiolar tissue.

A tumour that secretes glucagon-like peptide-1 and somatostatin in a patient with reactive hypoglycaemia and diabetes.

Glucagon-like peptide 1 (GLP-1), an insulinotropic hormone normally synthesised in the intestinal mucosa and released in response to a meal, is essential for normal glucose homoeostasis. There is much interest in the use of GLP-1 to treat diabetes, since the risk of hypoglycaemia is thought to be low. We report an instance of a 45-year-old woman with a GLP-1 and somatostatin secreting neuroendocrine tumour who presented with reactive hypoglycaemia and hyperglycaemia, but who was subsequently cured by surgery. This case, of a neuroendocrine tumour secreting GLP-1 and causing reactive hypoglycaemia, indicates a potential adverse effect of GLP-1 therapy for diabetes.

A quantitative study of the neural changes underlying pyloric stenosis in dogs.

This study aimed to quantify the neural changes in congenital pyloric stenosis in dogs and to study the comparative anatomy between this condition in dogs and that in infantile hypertrophic pyloric stenosis. Eight specimens from the pylorus of dogs with pyloric stenosis and six control specimens were examined using conventional histology and immunohistochemistry for a range of neural antigens. The changes in the proportion of nerves immunoreactive for each antigen were quantified and analysed statistically. The morphology of the nerves in the diseased dogs was similar to that in controls. Only vasoactive intestinal peptide was reduced in expression in dogs (median proportion in control dogs 0.57, in diseased dogs 0.17; P = 0.065). This study demonstrates both morphological similarities and significant differences between closely related conditions in dogs, humans and other species.

Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis.

Pulmonary arteries of patients with severe pulmonary hypertension (SPH) presenting in an idiopathic form (primary PH-PPH) or associated with congenital heart malformations or collagen vascular diseases show plexiform lesions. It is postulated that in lungs with SPH, endothelial cells in plexiform lesions express genes encoding for proteins involved in angiogenesis, in particular, vascular endothelial growth factor (VEGF) and those involved in VEGF receptor-2 (VEGFR-2) signalling. On immunohistochemistry and in situ hybridization, endothelial cells in the plexiform lesions expressed VEGF mRNA and protein and overexpressed the mRNA and protein of VEGFR-2, and the transcription factor subunits HIF-1alpha and HIF-1beta of hypoxia inducible factor, which are responsible for the hypoxia-dependent induction of VEGF. When compared with normal lungs, SPH lungs showed decreased expression of the kinases PI3 kinase and src, which, together with Akt, relay the signal transduction downstream of VEGFR-2. Because markers of angiogenesis are expressed in plexiform lesions in SPH, it is proposed that these lesions may form by a process of disordered angiogenesis.

Pantoprazole therapy in the long-term management of severe acid peptic disease: clinical efficacy, safety, serum gastrin, gastric histology, and endocrine cell studies.

OBJECTIVE: Pantoprazole is the third proton pump inhibitor to become available. When this study was started, there were few data on its long-term use. Our aim was to investigate this aspect and, because powerful inhibitors of acid secretion can cause hypergastrinemia and, in experimental animals, enterochromaffin-like cell hyperplasia, we also monitored serum gastrin and endocrine cell histology. METHODS: One hundred fifty patients refractory to H2-receptor antagonists, running an aggressive course or with complications, were entered into a 5-yr treatment program. We performed serial endoscopy, checked for adverse events, and laboratory values. We also monitored serum gastrin, gastric endocrine cell histology, and antral and corpus gastritis. RESULTS: This report presents results from up to 3 yr of treatment. Cumulative healing on 40-80 mg of pantoprazole was 82% at 4 wk and 92% by 12 wk. Most patients became asymptomatic within 4 wk. Remission on maintenance treatment with 40 mg (n = 111) was 85% at 12 months and 78% at 24 months. Treatment was safe; only four patients had adverse events definitely related to pantoprazole. Elevations in gastrin were modest and there were no significant changes in gastric endocrine cells. The number of enterochromaffin-like cells tended to decrease. CONCLUSION: Pantoprazole is effective, safe, and does not seem to be associated with large increases in serum gastrin or alterations in gastric endocrine cells.

Tumor necrosis factor-alpha is expressed in donor heart and predicts right ventricular failure after human heart transplantation.

BACKGROUND-Myocardial failure is an important problem after heart transplantation. Right ventricular (RV) failure is most common, although its mechanisms remain poorly understood. Inflammatory cytokines play an important role in heart failure. We studied the expression of tumor necrosis factor (TNF)-alpha and other cytokines in donor myocardium and their relationship to the subsequent development of RV failure early after transplantation. METHODS AND RESULTS-Clinical details were obtained, and ventricular function was assessed by transesophageal echocardiography in 26 donors before heart retrieval. A donor RV biopsy was obtained immediately before transplantation, and each recipient was followed for the development of RV failure. Reverse transcriptase-polymerase chain reaction was performed to detect TNF-alpha, interleukin-2, interferon-gamma, and inducible nitric oxide synthase expression. Eight of 26 recipients (30.8%) developed RV failure. Seven of these 8 (87.5%) expressed TNF-alpha, but only 4 of the 18 (22.2%) who did not develop RV failure expressed TNF-alpha (P<0.005). As a predictor of RV failure, TNF-alpha mRNA had a sensitivity of 87.5%, a specificity of 83.3%, a positive predictive value of 70%, and a negative predictive value of 93.7%. Western blotting demonstrated more TNF-alpha protein in the myocardium of donor hearts that developed RV failure (658+/-60 versus 470+/-57 optical density units, P<0.05). Immunocytochemistry localized TNF-alpha expression to cardiac myocytes. Reverse transcriptase-polymerase chain reaction detected interferon-gamma in 2 (7.7%), interleukin-2 in 1 (3.8%), and inducible nitric oxide synthase mRNA in 1 (3.8%) of the 26 donor hearts, none of which developed RV failure. CONCLUSIONS-TNF-alpha expression in donor heart cardiac myocytes seems to predict the development of RV failure in patients early after heart transplantation.

Up-regulation of inducible nitric oxide synthase in fibroblasts parallels the onset and progression of fibrosis in an experimental model of post-transplant obliterative airway disease.

The main cause of mortality following lung transplantation is chronic rejection, manifesting morphologically as obliterative bronchiolitis (OB). It has been suggested that damage to the respiratory epithelium initiates proliferation of mesenchymal cells, leading to dense collagenous scarring in small airways. Inducible nitric oxide synthase (iNOS) is strongly expressed in the damaged epithelium in human OB, along with high levels of peroxynitrite, suggesting that endogenous NO mediates the epithelial destruction. To examine further the role of iNOS in this process, heterotopic airway implants were studied in rats, an acknowledged disease model. Specimens of iso- or allografted trachea, collected 3-60 days after implantation, were processed for histology and immunocytochemistry for iNOS and, as a marker of peroxynitrite formation, nitrotyrosine. In both iso- and allografts at the earliest stage (day 3), ischaemia was associated with severe epithelial damage or loss. These changes progressed until day 7 and were accompanied by strong expression of iNOS and nitrotyrosine in epithelial cells. In isografts, epithelial recovery was seen, with abundant iNOS immunoreactivity but little nitrotyrosine. In contrast, the epithelium in allografts did not regenerate and progressive inflammation and fibroproliferation occurred until complete obliteration of the tracheal lumen at day 60. The fibroproliferation was associated with changes in morphology of fibroblasts that were accompanied by alterations in their iNOS expression. iNOS immunoreactivity was dense in the plump fibroblasts of early lesions, in some cases as early as post-operative day 5, but very weak in elongated fibroblasts in totally occluded grafts. The intensity of immunoreactivity for nitrotyrosine corresponded to that of iNOS. These results indicate a dual role for NO in the airway obliteration that follows transplantation, through destruction of epithelium and stimulation of fibroblast activity.

Neutrophils enhance expression of inducible nitric oxide synthase in human normal but not cystic fibrosis bronchial epithelial cells.

The bronchial epithelium in cystic fibrosis (CF) expresses very low levels of the inducible form of nitric oxide synthase (iNOS). The product of iNOS, nitric oxide (NO), mediates anti-microbial effects and can reduce neutrophil sequestration in the lung. Heavy neutrophilic infiltration of the pulmonary epithelium is a major feature of the end-stage CF lung. This study hypothesized that the system whereby the pulmonary epithelium protects itself against exaggerated neutrophilic infiltration by producing NO is compromised in CF. Human neutrophils were activated by incubation with cytokines, added to monolayers of normal (16HBE14o-) and CF (CFBE41o-) bronchial epithelial cells and co-cultured for up to 72 h. Marked up-regulation of iNOS protein expression was seen in normal bronchial epithelial cells following neutrophil co-culture but the CF cells showed a significantly smaller increase (p<0.001). To determine whether the relative lack of protein was due to a defect in translation, RT-PCR of iNOS mRNA was carried out and a pattern of mRNA expression was seen paralleling that of the protein. The reduced production of NO by CF compared with normal epithelium was shown by the presence of significantly (p<0.001) less accumulated nitrites in medium after co-culture with neutrophils. In summary, this study shows that the normal production of NO by bronchial epithelium in response to contact with neutrophils is lacking in CF. As NO has been shown to oppose neutrophil sequestration, its relative lack in CF may underlie the heavy neutrophilic infiltration that characterizes the disease.