Maternal environmental enrichment protects neonatal brains from hypoxic-ischemic challenge by mitigating brain energetic dysfunction and modulating glial cell responses.

There is evidence that maternal milieu and changes in environmental factors during the prenatal period may exert a lasting impact on the brain health of the newborn, even in case of neonatal brain hypoxia-ischemia (HI). The present study aimed to investigate the effects of maternal environmental enrichment (EE) on HI-induced energetic and metabolic failure, along with subsequent neural cell responses in the early postnatal period. Male Wistar pups born to dams exposed to maternal EE or standard conditions (SC) were randomly divided into Sham-SC, HI-SC, Sham-EE, and HI-EE groups. Neonatal HI was induced on postnatal day (PND) 3. The Na+,K+-ATPase activity, mitochondrial function and neuroinflammatory related-proteins were assessed at 24 h and 48 h after HI. MicroPET-FDG scans were used to measure glucose uptake at three time points: 24 h post-HI, PND18, and PND24. Moreover, neuronal preservation and glial cell responses were evaluated at PND18. After HI, animals exposed to maternal EE showed an increase in Na+,K+-ATPase activity, preservation of mitochondrial potential/mass ratio, and a reduction in mitochondrial swelling. Glucose uptake was preserved in HI-EE animals from PND18 onwards. Maternal EE attenuated HI-induced cell degeneration, white matter injury, and reduced astrocyte immunofluorescence. Moreover, the HI-EE group exhibited elevated levels of IL-10 and a reduction in Iba-1 positive cells. Data suggested that the regulation of AKT/ERK1/2 signaling pathways could be involved in the effects of maternal EE. This study evidenced that antenatal environmental stimuli could promote bioenergetic and neural resilience in the offspring against early HI damage, supporting the translational value of pregnancy-focused environmental treatments.

Mild hyperhomocysteinemia alters extracellular adenine metabolism in rat brain.

Since homocysteine (Hcy) is considered a risk factor to cerebral diseases and adenine nucleotides are important molecules to brain normal function, in the present study we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in rat cerebral cortex. The levels of ATP, ADP, AMP and adenosine (Ado) in cerebrospinal fluid (CSF) of adult rats also were evaluated by high-performance liquid chromatography. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) was administered subcutaneously from the 30th to the 60th day of life. Control rats received saline solution in the same volumes. Results showed that Hcy significantly decreased nucleotide hydrolysis in the synaptosomal fraction and increased E-NTPDase1 and ecto-5'-nucleotidase transcripts in rat cerebral cortex. ATP levels were significantly increased, while Ado decreased in CSF of Hcy-treated rats. These findings suggest that the unbalance in ATP and Ado levels may be, at last in part, involved in the cerebral toxicity of mild hyperhomocysteinemia.

[Eye muscle surgery in unilateral abducens palsy]. / Augenmuskelchirurgie bei einseitiger Abduzensparese.

BACKGROUND: As there are only few data on squint angle reduction following surgical treatment of unilateral abducens palsy, we aimed to quantify squint angle reduction after several different surgical procedures. PATIENTS AND METHODS: Retrospective analysis of 88 consecutive files of patients with unilateral abducens palsy, treated in 2000 - 2007 (46 resections of the lateral rectus muscle, 25 resections of the lateral rectus combined with recession of the medial rectus, 17 Hummelsheim transpositions, modified by Kaufmann). Maximal abduction was possible up to primary position in all 17 patients with Hummelsheim transposition. All other patients (except two) were able to abduct beyond primary position. RESULTS: In resections of the lateral rectus a stable dose-effect-correlation was found: the dose-effect coefficients (DEC) ranged between 1.5 degrees and 1.6 degrees reduction of horizontal angle (far fixation)/mm of resected muscle. In combined convergence procedures the DEC ranged from 1.52 degrees /mm (7-9 mm recession/resection) up to 1.39 degrees /mm (13-15 mm recession/resection). In muscle transpositions (Hummelsheim-Kaufmann), preoperative horizontal squint angle (far distance) was reduced from +29 degrees (median, range +15 degrees to +50 degrees ) to -3 degrees (median, range -15 degrees to +17 degrees ) postoperatively (6-8 weeks). The best results were achieved with preoperative squint angels between > +20 degrees and < +35 degrees . Larger basic angles showed mostly undercorrection; smaller angles showed always overcorrection. CONCLUSIONS: Unilateral abducens palsy with maximal abduction up to primary position should be treated by muscle transposition. With squint angles (far distance) < +20 degrees a classical Hummelsheim transposition is recommended, with squint angles > +20 degrees the Kaufmann's modification should be preferred. If abduction beyond primary position is possible, lateral rectus resection suffices. With squint angles > +12 degrees additional recession of the ipsilateral medial rectus muscle becomes necessary.

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-kappa B inhibition.

Addressing the puzzling role of amidated gastrin(17) (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappaB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappaB activation and decreased expression of the antiapoptotic NF-kappaB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFalpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappaB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFalpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFalpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappaB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

Genetic deletion of JNK1 and JNK2 aggravates the DSS-induced colitis in mice.

The c-Jun N-terminal kinases (JNKs) are considered as novel targets for therapy of inflammatory bowel diseases (IBD). However, the relevant JNK isoforms have to be elucidated. Here, we analyze the individual contribution of the JNK1 and JNK2 isoforms in a dextran sulfate sodium (DSS) model of experimental colitis. JNK1 and JNK2 knockout mice (JNK1 ko, JNK2 ko) and their wild-type controls (WT1, WT2) received three cycles of DSS treatment, each consisting of 1.7% DSS for 5 days, followed by 5 days with water. Animals were daily evaluated by a disease activity index (DAI) comprising measurement of body weight, estimation of stool consistency, and test for occult blood/gross rectal bleeding. After 30 days all animals were sacrificed, and the inflamed intestine was histologically evaluated by a crypt damage score. Unexpectedly, neither JNK1 ko nor JNK2 ko prevented mice from developing a chronic colitis when compared to wild-type controls WT1 and WT2, respectively. On the contrary, DAI and mortality were aggravated in JNK2 ko compared to WT2. DAI and mortality did not differ between JNK1 ko and WT1, but the histological crypt damage score was significantly enhanced in the cecum of JNK1 ko mice. Genetic deletion of JNK2 worsens the disease outcome in an experimental model of murine colitis. We hypothesize that the functional deletion of the otherwise proapoptotic JNK2 prolongs the activity of proinflammatory immune cells with deterioration of disease activity.

L-amino acid load to enhance PET differentiation between tumor and inflammation: an in vitro study on (18)F-FET uptake.

Labeled amino acids (AA) are tumor tracers for use in nuclear medecine. O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET) is transported by the L-system, known to function as an exchanger. In vitro utilization of FET, after a preload or prior to an afterload of non radioactive L-amino acids, was evaluated in order to measure the potential effects of AA content on the distinction between tumor and inflammatory lesions. Cellular uptake of FET was studied on rat osteosarcoma cells (ROS 17/2.8) and human leukocytes, initially loaded with nonradioactive L-tyrosine or L-methionine. FET efflux was evaluated from cells loaded with nonradioactive L-phenylalanine after tracer uptake. ROS 17/2.8 showed a higher sensitivity to preload and afterload effects on cellular FET content as compared with the leukocytes. We conclude that preload with L-tyrosine, prior to the administration of FET, may be a potential procedure to improve PET differentiation between tumor and inflammatory lesions.

Vascular endothelial growth factor (VEGF164) ameliorates intestinal epithelial injury in vitro in IEC-18 and Caco-2 monolayers via induction of TGF-beta release from epithelial cells.

OBJECTIVE: VEGF is a glycoprotein with various (e.g. angiogenic) activities. So far, research has focused on its angiogenic properties. VEGF receptors are localized on epithelial cells of patients with inflammatory bowel disease (IBD) and also on Caco-2 and IEC-18 cells. Our aim was to evaluate the role of VEGF on intestinal epithelial cell (IEC) migration and proliferation by utilizing an established in vitro model. METHODS: IEC-18 and Caco-2 monolayers were wounded with a razor blade as described previously. Cells were incubated in medium w/o rat VEGF(164). After 24 h, migration was assessed by counting cells across the wound edge. Migration was blocked with neutralizing TGF-beta(1) antibodies. IEC proliferation was assessed using the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) test. Semi-quantitative changes of the TGF-beta(1) mRNA expression were evaluated before and after stimulation of the cells with VEGF(164) by RT-PCR. Statistical analysis was performed with ANOVA and the Wilcoxon test. RESULTS: VEGF(164) significantly induced epithelial cell migration in Caco-2 and IEC-18 cells compared to control. TGF-beta(1) antibodies completely abolished this VEGF-induced cell migration. TGF-beta(1) mRNA significantly increased in IEC-18 and Caco-2 cells after stimulation with VEGF. VEGF significantly inhibited epithelial cell proliferation in IEC-18 and in Caco-2 cells, indicating that the observed effects on cell migration were not due to any proliferate effects. CONCLUSION: VEGF effects on epithelial cell migration play an important part in epithelial cell restitution by maintaining mucosal homeostasis after mucosal injury. This effect is mediated by TGF-beta(1). Our results obtain another possible role for increased VEGF levels in the intestinal mucosa of patients with IBD as reported recently by others.

Mutations in the calcium-sensing receptor: a new genetic risk factor for chronic pancreatitis?

OBJECTIVE: In 2003 we identified a family with familial hypocalciuric hypercalcemia (FHH) (heterozygous CASR gene mutation L173P) and a mutation in the pancreatic secretory trypsin inhibitor gene (SPINK1) (N34S). While family members with an isolated calcium-sensing receptor gene (CASR) mutation remained healthy, a combination of the CASR and SPINK1 gene mutation caused chronic pancreatitis (CP). We thus speculate that the combination of two genetic defects affecting calcium homeostasis and pancreatic enzyme activation might represent a novel approach in chronic inherited pancreatic disease. We therefore sought to explore whether CASR gene mutations were prevalent in a cohort of patients with CP and confirmed SPINK1 mutations. MATERIAL AND METHODS: A cohort of 19 families (n=170) with a history of idiopathic CP (ICP) was screened for mutations within the CASR gene; 104 members of that cohort had a mutation (N34S) within the SPINK1 gene and 66 of those were suffering from CP. The entire CASR gene was screened for single strand conformation polymorphism under varying polyacrylamide gel conditions and subjected to direct dideoxy nucleotide sequencing of amplified cDNA. RESULTS: Single-strand conformation polymorphisms were observed in 59 samples, clustering of exons 3, 4 and 7. DNA sequence analysis revealed a yet unreported missense mutation in exon 7 (R896H) and two conservative mutations in exon 4 (F391F) and exon 7 (E790E). Furthermore, an intronic polymorphism in nucleotide position 493-19 G>A was detected in 19 out of 170 members of that cohort. CONCLUSIONS: We identified three novel calcium-sensing receptor gene mutations (1 missense mutation, 2 silent mutations and 1 intronic polymorphism) in a cohort of 19 families with ICP. In particular, the kindred with the R896H mutation presenting with a similar pedigree to the family described above may indicate a role for CASR gene mutations in SPINK1-related CP. Again, only the patient with the combination of both CASR and N34S SPINK1 gene mutation developed pancreatitis, whereas in the healthy parents and children only an isolated CASR or N34S SPINK1 gene mutation could be detected. We suggest that the CASR gene is a novel yet undetected co-factor in a multifactorial genetic setting of SPINK1-related pancreatitis that alters the susceptibility for pancreatitis in these patients.

Glucagon-like peptide 2 improves intestinal wound healing through induction of epithelial cell migration in vitro-evidence for a TGF--beta-mediated effect.

BACKGROUND/AIMS: In vitro studies suggest that glucagon-like peptide 2 (GLP-2), secreted from enteroendocrine cells in the gastrointestinal tract after food intake, is able to ameliorate mucosal injury in settings of human disease characterized by injury and dysfunction of the intestinal mucosal epithelium. We evaluated this potential of GLP-2 after epithelial trauma by using two in vitro models measuring intestinal epithelial cell proliferation and cell migration. MATERIALS AND METHODS: Injuries were induced in confluent monolayers of the small intestinal cells lines IEC-6 and IEC-18, as well as in the colonic cell lines Caco-2 and Colo 320. GLP-2 (50-500 nM) or other peptides were added to the media. Wound healing was investigated after 24 h by quantification of the number of cells migrating across the wound edge. Proliferation of cells was assessed by using photometric mitochondrial incorporation measurement of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Monoclonal TGF-beta antibodies were added to wounded monolayers to examine whether the GLP-2-induced wound healing was TGF-beta-mediated. RESULTS: Migration assessments revealed a significant stimulation of GLP-2-induced migration in IEC-6 and IEC-18 monolayers compared to the placebo group. No effect was observed in the colon cancer cell lines Caco-2 and Colo 320. Results of the proliferation assays show a significant inhibition of proliferation by GLP-2 in small intestinal cell lines whereas a dose-dependent stimulation of proliferation in colonic epithelial cells was observed. Addition of neutralizing TGF-beta1 antibodies to wounded IEC-6 and IEC-18 monolayers incubated with GLP-2 significantly reduced the number of migrating cells to the level of the placebo group. CONCLUSIONS: In our in vitro model, it was shown that the GLP-2-induced improvement of intestinal wound healing is TGF-beta-mediated. These effects were predominant in the epithelium of the small intestine compared to colonic epithelium. Our findings provide further insight into mechanisms leading to GLP-2-induced mucosal wound healing. These results suggest that GLP-2 or analogues of this peptide may potentially be useful for the treatment of intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

Acquired pure megakaryocytic aplasia: a separate haematological disease entity or a syndrome with multiple causes?

We report the case of a patient with acquired pure megakaryocytic aplasia. Until today, less than 20 cases of acquired pure megakaryocytic aplasia have been reported and the disease aetiology still seems to be unclear. This report summarizes the published data concerning possible aetiologies, treatment options and outcome of patients with acquired pure megakaryocytic aplasia. Furthermore, this case report presents an example for a possible disease progression.

The interleukin-6 promoter polymorphism is associated with elevated leukocyte, lymphocyte, and monocyte counts and reduced physical fitness in young healthy smokers.

Smoking and interleukin-6 are important factors in driving inflammation. This study assessed the relationship between smoking, interleukin-6 genotype, physical fitness, and peripheral blood count in healthy young men. For this interleukin-6 promoter polymorphism -174 genotype-phenotype association study 1,929 healthy German male aviators recruited at the central German Air Force Institute of Aviation Medicine were stratified by smoking habits. Cardiovascular fitness was expressed as maximal physical working capacity (PWCmax) in watts per kilogram body weight as assessed by maximal exercise testing by cycle ergometry up to physical exhaustion. Smokers had higher leukocyte and lymphocyte counts than nonsmokers and lower PWCmax. In the overall study population the C allele of the interleukin-6 polymorphism was weakly associated with elevated leukocytes and lymphocytes; in nonsmokers the interleukin-6 polymorphism was not associated with altered phenotypes, but in smokers the interleukin-6 C allele was associated with higher leukocytes, lymphocytes, and monocytes and with lower PWCmax. Smoking is thus associated with elevated leukocytes and lymphocytes and with reduced physical fitness. Gene carriers with the interleukin-6 C allele may suffer particularly from cigarette smoking.

Cardiovascular risk factors in patients with aortic stenosis predict prevalence of coronary artery disease but not of aortic stenosis: an angiographic pair matched case-control study.

BACKGROUND: Traditional cardiovascular risk factors have been associated with aortic stenosis and coronary artery disease. As these two conditions often co-exist, the association of cardiovascular risk factors with aortic stenosis may reflect confounding. OBJECTIVE: To compare the cardiovascular risk profile in patients with severe aortic stenosis undergoing elective coronary angiography with that of patients without aortic stenosis or calcification undergoing coronary angiography for suspected coronary artery disease. METHODS: 523 patients referred for elective diagnostic left heart catheterisation because of severe aortic stenosis formed the case population; 3925 patients without valve disease referred for elective diagnostic left heart catheterisation formed the base control population. Of the latter, 523 were pair matched to the case population for sex, age, and prevalence of relevant coronary artery disease, forming a pair matched control population. Cardiovascular risk factors (male sex, hypertension, hypercholesterolaemia, smoking, diabetes mellitus, family history of coronary artery disease) were assessed in all the patients. RESULTS: None of the cardiovascular risk factors was more prevalent in patients with aortic stenosis than in the base control population or in the pair matched control population. However, male sex, hypercholesterolaemia, smoking, diabetes mellitus, and a family history of coronary artery disease were significantly associated with the presence of additional coronary artery disease in patients with aortic stenosis. CONCLUSIONS: Cardiovascular risk factors are commonly present in patients with aortic stenosis. However, when compared with controls matched for age, sex, and angiographically defined coronary artery disease, no risk factor was significantly associated with the prevalence of aortic stenosis. Thus other factors are likely to be more important in the pathogenesis of aortic stenosis.

The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells.

BACKGROUND: Acetylsalicylic acid (ASA, aspirin), the most common nonsteroidal anti-inflammatory drug (NSAID), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the mechanism of its anticancer function remains unclear. The aim of this study was to determine the effects of acetylsalicylic acid on proliferation, apoptosis, and invasion in human cyclooxygenase-2 (COX-2) negative colorectal cancer cell lines. MATERIALS AND METHODS: After treatment with various concentrations of ASA, cell proliferation was measured in the human colon cancer cell line SW480. Apoptotic cells were identified by transmission electron microscopy, acridine orange staining, and flow cytometry. The invasive potential of SW480 cells was detected using an in vitro invasion assay. The production of carcinoembryonic antigen was measured by microparticle enzyme immunoassay. Expression of Bcl2, Bax, CD44v6, and nm23 were evaluated by immunocytochemistry. RESULTS: ASA significantly inhibited the proliferation of SW480 cells and stimulated apoptosis. Production of carcinoembryonic antigen and the invasive potential of SW480 cells were also inhibited by ASA. After treatment with ASA, down-regulation of Bcl2 and CD44v6 expression and up-regulation of nm23 expression were observed in SW480 cells. No obvious effect of ASA was found on Bax expression. CONCLUSION: Our findings reveal that ASA inhibits the proliferation and promotes apoptosis in the human colon cancer cell line SW480. Down-regulation of Bcl2 expression might represent a potential mechanism by which ASA induces apoptosis in this COX-2 negative colon cancer cell line. Our results also suggest that ASA decreases the invasive potential of these colon cancer cells. Decreased CEA content and CD44v6 expression and elevated nm23 expression may contribute to the effect of ASA on invasive potential of SW480 colon cancer cells.

[The nuclear medicine department and the TEP/Biomedical Cyclotron unit]. / Le Service de Médecine Nucléaire et l'Unité TEP/Cyclotron Biomedical.

During the last 25 years, the clinical and experimental activity in nuclear medicine at Erasme hospital has been influenced by the implementation of positron emission tomography (PET) in 1990 as a method of brain functional investigation. The activity of the PET/biomedical cyclotron unit has been dedicated to various subjects in neurology, neurosciences, psychiatry, oncology and cardiology. This has been made possible by developments in radiochemistry. The radiochemistry laboratory has designed and produced original tracers such as 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)-methyl]guanine (FHPG), a tracer of viral thymidine kinase activity in gene therapy protocols. We have brought new applications of PET, such as its integration into stereotactic neurosurgical and radioneurosurgical techniques in order to improve their diagnostic and therapeutic performance in neurooncology. We have also conducted multiple studies on brain physiology and pathophysiology, in particular with the use of functional and metabolic brain mapping methods and the use of tracers of neurotransmission systems. The Department of nuclear medicine has also performed studies on bone metabolism and investigated in vivo imaging methods of infectious and immune processes.

Expression of hepatocyte growth factor, keratinocyte growth factor and their receptors in experimental chronic pancreatitis.

BACKGROUND: Hepatocyte (HGF) and Keratinocyte growth factors (KGF) are key factors of tissue organization and regeneration. These peptide growth factors and their receptors c-met and keratinocyte growth factor receptor (KGFR) are overexpressed in pancreatic cancer. AIM: Expression and localization of ligands and receptors were investigated during the development of experimental chronic pancreatitis. METHODS: Chronic pancreatitis was induced in rats by intravenous injection of dibutyltin dichloride. One to 60 days after treatment, the expression of growth factors and receptors was analysed by competitive polymerase chain reaction, Western blot analysis and immunohistochemistry. RESULTS: HGF mRNA expression increased (10-fold) until days 7-14 followed by a decrease to control level. Expression of c-met mRNA constantly increased (15-fold). KGF and KGFR mRNA expression were increased after 14-28 days (5-fold) and then returned to control levels. mRNA expression patterns correlated with changes in the protein expression, whereas protein levels of KGF remained unchanged. Ligands were localized in mesenchymal cells and their receptors on epithelial cells. CONCLUSIONS: The significant increase of HGF and c-met expression suggests an essential role of this growth factor in the morphological changes during the development of chronic pancreatitis. Changes in the expression of KGF and KGFR are less pronounced.

Cellular expression of CCK-A and CCK-B/gastrin receptors in human gastric mucosa.

Gastrin stimulates gastric acid secretion in various species, but the role of the structurally related CCK for the peripheral regulation of acid secretion in humans remains controversial. Moreover, species differences in CCK receptor function and expression have been reported. We therefore sought to identify the cellular targets of CCK and gastrin within the human gastric mucosa in situ. Gastric biopsies were collected from 15 patients without gastric disease. Expression of CCK receptor subtypes was detected in individual cells of the gastric mucosa by reverse transcription (RT)-PCR in situ, immunohistochemistry and confocal laser scanning microscopy, using antisera against the CCK-A or CCK-B/gastrin receptor subtype. Both CCK-A and CCK-B receptors were detected in antral and oxyntic mucosa at the mRNA and protein level. In fundic mucosa, CCK-A receptor mRNA and protein mapped to D cells (37.4+/-7.7). Besides, individual chief cells, mucous neck cells and parietal cells (12.3+/-4.7%) expressed CCK-A receptors. CCK-B/gastrin receptor mRNA and protein were detected in parietal cells (57.4+/-11.1%) and in neuroendocrine cells (33.2+/-4.4%) expressing chromogranin A. Furthermore, epithelial cells within the neck of the gastric gland were found to express the CCK-B/gastrin receptor. We conclude that (i) identification of CCK-A receptors on somatostatin producing D cells in humans provide the anatomical basis for a receptor-mediated mode of action of CCK on somatostatin release and (ii) detection of either CCK receptor subtype in the putative stem cell compartment implies a role of CCK in the maintenance of tissue homeostasis in human gastric mucosa.

CCK-B/gastrin receptors in human colorectal cancer.

BACKGROUND: Mature amidated gastrin (G17 amide) mediates its effects in the gastrointestinal tract by activating G protein-coupled CCK-B/gastrin receptors. Although trophic actions of gastrin on the gastric mucosa have been well-established, the effect of G17 amide, progastrin and intermediates to colon neoplasia in humans is controversial. While epidemiological evidence from patients with elevated serum gastrin levels related to pernicious anaemia does not support an increased risk for colon cancer, a recent study suggests that prolonged hypergastrinaemia is associated with an increased risk for colon cancer. The extent to which trophic actions of gastrin in colorectal cancer are mediated by functional gastrin receptors remains to be defined. The aim of the present study was to determine CCK-B/gastrin receptor expression, structure, and function in 79 patients with colon cancer. MATERIALS AND METHODS: CCK-B/gastrin receptor cDNAs were isolated from 79 human colorectal cancer specimens and 15 control tissues, subcloned into the eukaryotic expression vector pCR3.1 and subjected to DNA sequence analysis. Wild-type and mutant cDNAs were transiently expressed in COS-7 cells to determine ligand affinities by 125I-labelled CCK-8S competition binding. Activation of the MAP kinase signalling cascade by G17 amide was determined in transfected Colo 320 cells expressing the wild-type or mutant CCK-B/gastrin receptors. Clonal expansion of single cells was quantified in transfected Colo 320 cells. RESULTS: Gastrin mRNA is expressed in 44% of colorectal cancers and in 13% of control tissues. CCK-B/gastrin receptor mRNA is expressed in 38% of colorectal cancers and 13% of normal colonic tissue. Co-expression of gastrin and CCK-B/gastrin receptor message is significantly increased in colorectal cancer specimens (32% vs. 0%). There is no correlation between CCK-B/gastrin receptor expression and disease stage or histological grading. DNA sequence analysis revealed one spontaneous CCK-B/gastrin receptor mutation within the third intracellular loop with an exchange of valine-287 for phenylalanine. Pharmacological characterisation of the 287V --> F CCK-B/gastrin receptor reveals wild-type affinities for G17 amide, glycine-extended gastrin, CCK-8S and L-365,260. Mutation 287V --> F is associated with a loss of gastrin-induced MAPK p44/p42 signalling in Colo 320 cells while clonal expansion from single cells is increased by 53.1 +/- 15.9% when compared to Colo 320 cells expressing wild-type CCK-B/gastrin receptors. CONCLUSIONS: Structural alterations of CCK-B/gastrin receptors may account for increased growth-promoting effects of amidated gastrins in colorectal cancer.

Identification of CCK-B/gastrin receptor splice variants in human peripheral blood mononuclear cells.

There is increasing evidence for a direct interaction of the enteric nervous and immune system. Receptors for neuropeptides such as VIP, somatostatin, and substance P have been characterised in human immuno-haematopoietic cells but little is known about the functional significance and expression of receptors for cholecystokinin (CCK) on cells of the immune system. There are only few studies that describe the expression of CCK receptors on human leukaemia-derived cell lines but the receptor structure and function in normal leukocytes have not been clearly established. We therefore sought to determine CCK receptor expression, structure, and function in nontransformed human peripheral blood mononuclear cells.Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in peripheral blood mononuclear cells from healthy volunteers without haematopoietic malignancy. In addition to wild-type CCK-B/gastrin receptor cDNAs, we isolated a splice variant with an in frame insertion of 69 amino acids within its putative third intracellular receptor loop. Dideoxy sequence analysis revealed that the cDNA of this splice variant comprises exons 1-4 but retains intron 4 (207 bp) in the absence of mutations within the splice donor sites. Transient expression of this splice variant in COS-7 cells reveals wild-type affinity for CCK-8, Gastrin-17, and antagonist L-365,260. Affinity for glycine-extended gastrin-17 was not increased when compared to the wild-type CCK-B/gastrin receptor. In vitro, gastrin decreased 3H-thymidine labelling in phytohaemagglutinin-pretreated mononuclear cells at a half-maximally effective concentration of 1.5 nM. We also isolated a cDNA encoding another splice variant of the CCK-B/gastrin receptor with a 158 bp deletion of the entire exon 4 sequence. We conclude that wild-type transcripts of both CCK receptor subtypes and splice variants of the CCK-B/gastrin receptor are expressed in nontransformed human mononuclear cells and that gastrin exhibits antiproliferative effects.