The use of a synthetic prostaglandin e1 analogue (misoprostol) as an adjunct to pancreatic enzyme replacement in cystic fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2% p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

The use of a synthetic prostaglandin-E1 analog (misoprostol) as an adjunct to pancreatic-enzyme replacement in cystic-fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2%, p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption.

Neonatal screening for cystic fibrosis

Two groups of patients with cystic fibrosis were compared. The screened group, detected with an improved neonatal screening assay for immunoreactive trypsin, developed fewer chest infections requiring treatment and gained more weight than the unscreened group. Early diagnosis by screening seems to affect early morbidity.

Serum immunoreactive cationic trypsinogen: A useful indicator of severe exocrine dysfunction in the paediatric patient without cystic fibrosis

We evaluated serum cationic trypsinogen as a marker of exocrine pancreatic function in children without cystic fibrosis. The ability of this test to determine steatorrhoea of pancreatic origin, and its relationship to a wide range of exocrine pancreatic function were assessed. Serum trypsinogen was measured in 32 children with steatorrhoea, 10 with pancreatic and 22 with non-pancreatic causes. In patients with pancreatic steatorrhoea, serum cationic trypsinogen was 4·9±4·9 μg/l (mean ±SD), significantly below values in patients with non-pancreatic steatorrhoea (47·0±22·1 μg/l, p<0·001) and 50 control subjects (31·4±7·4 μg/l, p<0·001). Serum cationic trypsinogen values in patients with pancreatic steatorrhoea all fell below the lower limit of our control range and below all values for patients with non-pancreatic steatorrhoea. Serum cationic trypsinogen was also evaluated against pancreatic trypsin output in 47 patients (range 0·2-17·0 yr) who underwent a hormonal pancreatic stimulation test. In 17 patients, serum cationic trypsinogen was low (<-2SD or 16·6 μg/l), and associated with greatly impaired pancreatic trypsin output, ranging from 0-8% of mean normal trypsin output. Five of these 17 patients did not have steatorrhoea. In 30 patients with normal or raised serum cationic trypsinogen (≥16·6 μg/l), pancreatic trypsin output ranged from 15-183% of mean normal values. In conclusion, low serum cationic trypsinogen suggests severely impaired exocrine pancreatic function, with sensitivity extending above the steatorrhoeic threshold. In the presence of steatorrhoea, low serum cationic trypsinogen indicates a pancreatic aetiology. Normal serum cationic trypsinogen, however, does not exclude impaired pancreatic function, above the steatorrhoeic threshold.

Age-related alterations of immunoreactive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic insufficiency

Serum immunoreactive cationic trypsinogen levels were determined in 99 control subjects and 381 cystic fibrosis (CF) patients. To evaluate the status of the exocrine pancreas all CF patients had previously undergone fecal fat balance studies and/or pancreatic stimulation tests. Three hundred fourteen CF patients had fat malabsorption and/or had inadequate pancreatic enzyme secretion (pancreatic insufficiency) requiring oral pancreatic enzyme supplements with meals. Sixty-seven CF patients did not have fat malabsorption and/or had adequate enzyme secretion (pancreatic sufficiency) and were not receiving pancreatic enzyme supplements with meals. Mean serum trypsinogen in 99 control subjects was 31.4 ± 14.8 /µg/hter (± 2 SD) and levels did not vary with age or sex. In CF infants (< 2 yr) with pancreatic insufficiency, mean serum trypsinogen was significantly above the non-CF values (p < 0.001). Ninety-one percent of the CF infants had elevated levels. Serum trypsinogen values in the pancreatic insuffi ient group declined steeply up to 5 years, reaching subnormal values by age 6. An equation was developed which described these age-related changes very accurately. Only six CF patients with pancreatic insufficiency had serum trypsinogen levels above the 95% confidence limits of this equation. In contrast, there was no age related decline in serum trypsinogen among the CF group with pancreatic sufficiency. Under 7 yr, serum trypsinogen failed to distinguish the two groups. In those over 7 yr of age, however, serum trypsinogen was significantly higher than the CF group with pancreatic insufficiency (p < 0.001), and 93% had values within or above the control range. In conclusion, serum trypsinogen appears to be a useful screening test for CF in infancy. Between 2 and 7 yr of age this test is of little diagnostic value. After 7 yr of age, serum trypsinogen can reliably distinguish between CF patients with and without pancreatic insufficiency.

Treatment of distal intestinal obstruction syndrome in cystic fibrosis with a balanced intestinal lavage solution

Conventional treatment of distal intestinal obstruction syndrome (DIOS) with high doses of pancreatic enzymes, mucolytic agents, and enemas is neither predictably effective nor rapid in action. In 6 cystic fibrosis patients with DIOS a balanced, non-absorbable intestinal lavage solution produced clinical and radiological improvement and striking improvement in DIOS scores. It is suggested that a balanced intestinal lavage solution should be considered as an alternative treatment for DIOS in patients with cystic fibrosis.

Serum immunoreactive pancreatic lipase and cationic trypsinogen for the assessment of exocrine pancreatic function in older patients with cystic fibrosis

Indirect and qualitative tests of pancreatic function are commonly used to screen patients with cystic fibrosis for pancreatic insufficiency. In an attempt to develop a more quantitative assessment, we compared the usefulness of measuring serum pancreatic lipase using a newly developed enzyme-linked immunosorbent immunoassay with that of cationic trypsinogen using a radioimmunoassay in the assessment of exocrine pancreatic function in patients with cystic fibrosis. Previously, we have shown neither lipase nor trypsinogen to be of use in assessing pancreatic function prior to 5 years of age because the majority of patients with cystic fibrosis in early infancy have elevated serum levels regardless of pancreatic function. Therefore, we studied 77 patients with cystic fibrosis older than 5 years of age, 41 with steatorrhea and 36 without steatorrhea. In addition, 28 of 77 patients consented to undergo a quantitative pancreatic stimulation test. There was a significant difference between the steatorrheic and nonsteatorrheic patients with the steatorrheic group having lower lipase and trypsinogen values than the nonsteatorrheic group (P < .001). Sensitivities and specificities in detecting steatorrhea were 95% and 86%, respectively, for lipase and 93% and 92%, respectively, for trypsinogen. No correlations were found between the serum levels of lipase and trypsinogen and their respective duodenal concentrations because of abnormally high serum levels of both enzymes found in some nonsteatorrheic patients. We conclude from this study that both serum lipase and trypsinogen levels accurately detect steatorrhea in patients with cystic fibrosis who are older than 5 years but are imprecise indicators of specific pancreatic exocrine function above the level needed for normal fat absorption.

Age-related alterations in immunoreactive pancreatic lipase and cationic trypsinogen in young children with cystic fibrosis

Serum immunoreactive pancreatic lipase and cationic trypsinogen are elevated in young infants with cystic fibrosis (CF) and may be useful neonatal screening tests for CF. We compared lipase measured by a recently developed ELISA immunoassay with trypsinogen measured by radioimmunoassay in 70 children (ages 0.1 to 9.9 years) with CF who had various degrees of pancreatic dysfunction and in 79 similarly aged children without CF (controls). In the control children, lipase activity increased with advancing age, whereas trypsinogen showed no age-related trend. Lipase and trypsinogen were significantly elevated in the infants with CF who were younger than 1 year, irrespective of pancreatic function (trypsinogen, P<0.001; lipase, P<0.05). Sensitivities in detecting CF were 76% and 90% for lipase and trypsinogen, respectively. After the first year of life, lipase and trypsinogen values declined toward normal, the rate of decline of lipase being greater than that of trypsinogen; 67% of lipase values were within or below the normal range by 3 years, whereas 67% of trypsinogen values continued to be elevated. We conclude that trypsinogen is an excellent screening test for CF in young infants regardless of pancreatic function, and that the addition of a serum pancreatic lipase determination does not improve the accuracy of trypsinogen as a screening test for cystic fibrosis.

Quality of life changes during conformal radiation therapy for prostate carcinoma

BACKGROUND: The objective of this study was to describe prospectively quality of life (QOL) before and after radiotherapy for patients with prostate carcinoma. METHODS: Forty-three patients with T1-T3 prostate carcinoma who underwent conformal external beam radiation therapy were randomized either to the complete European Organization for Research and Treatment of Cancer (EORTC) QOL questionnaire (EORTC QLQ-C30) or the Medical Outcomes Study Group Short Form Health Survey (SF-36) at baseline, at 3 weeks and 6 weeks after initial treatment, and at 6 weeks and 5 months after the completion of radiotherapy. The measures were self-reported patient QOL, and values are given as the mean +/- standard error of the mean. Changes in QOL are described from baseline to the end of treatment in both questionnaire groups. RESULTS: Emotional role functioning, as measured with the SF-36 questionnaire, significantly improved from 68.2 +/- 9.9 at baseline to 93.3 +/- 5.2 at the end of therapy (P = 0.02). The EORTC QLQ-C30 questionnaire revealed consistent values of emotional functioning during treatment (72.7 +/- 5.9 at baseline) but showed a significant improvement 6 weeks after therapy (89.0 +/- 4.4; P = 0.01). Role functioning deteriorated from 80.1 +/- 6.5 at baseline to 62.5 +/- 8.8 at the end of radiotherapy (P = 0.02). Symptoms of fatigue were shown to increase significantly from 26.9 +/- 6.0 at baseline to 37.7 +/- 7.6 at the end of therapy (P = 0.02). No significant changes in the other dimensions were observed in either questionnaire. CONCLUSIONS: After radiotherapy for prostate carcinoma, patients experience a temporary deterioration of fatigue and role functioning, as measured with the EORTC QLQ-C-30. Despite physical deterioration, the authors observed an improvement in emotional functioning scores with both questionnaires. This may have been due to psychological adaptation and coping.

Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.