Gastrin-induced release of calcitonin from medullary thyroid carcinomas (MTC) is based on the expression of the cholecystokinin(2)-receptor (CCK(2)R) in these tumors. Recently, we have shown that the CCK(2)R is expressed not only in MTC but also in C-cells within the normal thyroid gland. The functions of the CCK(2)R in MTC and C-cells are largely unknown. We therefore explored the effects of gastrin-induced CCK(2)R stimulation in the highly differentiated MTC cell line, TT. CCK(2)R expression in TT-cells is detectable by RT-PCR as well as immunocytochemistry. Stimulation of the CCK(2)R by gastrin induces immediate release of calcitonin from TT-cells. Moreover, quantitative (LightCycler) RT-PCR demonstrates that gastrin stimulates transcription of the calcitonin and chromogranin A genes in TT-cells. TT-cell proliferation, assessed by counting of viable cells and (3)H-thymidine uptake, is markedly increased by gastrin. This effect is inhibited by the CCK(2)R-specific antagonist L-365,260. Our findings suggest physiological functions for the CCK(2)R in calcitonin-secretion and gene expression as well as a pathophysiological role in MTC proliferation. CCK(2)R antagonists might have therapeutic potential in these tumors.

OBJECTIVE:The cholecystokinin(2)-receptor (CCK(2)R) promotes secretion and cell growth induced by its ligands cholecystokinin (CCK) and gastrin. The receptor has recently been shown to be expressed in human medullary thyroid carcinomas (MTCs). The objective of this study was to analyze CCK(2)R expression in MTC samples of different tumor stages as well as in non-malignant thyroid tissues.DESIGN AND METHODS:Using RT-PCR we investigated 19 MTC samples and TT-cells (a human MTC cell line), as well as samples of normal thyroid. In addition, we performed immunohistochemistry using calcitonin- and CCK(2)R-specific antibodies on MTCs and samples of C-cell hyperplasia.RESULTS:We demonstrate for the first time that CCK(2)R is expressed not only in MTCs but in all samples of normal thyroid tissue. Using immunohistochemistry the receptor could be localized on calcitonin-secreting C-cells. The highest incidence of CCK(2)R expression in MTCs was observed in early-tumor stages, whereas CCK(2)R could not be detected in advanced or metastasized tumors.CONCLUSIONS:The expression of CCK(2)R in C-cells suggests a physiological function for gastrin and/or CCK in the regulation of calcitonin release, presumably related to bone and calcium metabolism. Moreover, these ligands might act as growth factors in MTCs. Efforts in the development of CCK(2)R scintigraphy for the detection of MTC lesions might have to consider a lower incidence of the receptor in advanced tumor stages.

Antimicrobial peptides (defensins) are a recently discovered component of the innate immune system. Human beta defensins (hBD1/hBD2), two novel members of the family of defensins, contribute to the antimicrobial defence of mucosal tissues like the lung, urogenital tract and the gastrointestinal tract. Both are either constitutively and inducible expressed. We have previously demonstrated widespread beta-defensiu expression in the human gastrointestinal tract. Furthermore one member of the family (hBD2) was shown to be upregulated after stimulation of proinflammatory cytokines. Aim: To study the physiological role of hBD1 and hBD2 in patients with Crohn's disease. Material and Methods: A) The influence of human serum from Crohn's patients with active disease (CD-Serum), or controls without apparent disease, on hBD1 and hBD2 expression was tested in a CaCo-2 cell assay. After extraction of total cell RNA of the stimulated cell lines a quantitative RT-PCR-analysis was performed with hBD1 and hBD2-specific-oligonucleotides. The following oligonucleotides were used: (hBD1) 5'-CCA GCC ATC AGC CAT GAG GGT-3'sense/3'GGA GCC cTr TCT GAA TCC GCA3'and (hBD2) 5'-CCC AGC CAT GAG GAC CAT CG-3'and 5'-6TCT ATC TAG GAA GCT CAG CG-3'. B) RNAs from human intestinal tissue (surgical specimens after partial culectomy or small bowel resection from Crohn's patients; n = 10) were evaluated for,8-dutensin expression using the described RT-PCR analysis. Results: A) Human colon epithelial cell lines express human/~--defensins. The expression of ,8,--defensin-1 (hBD1) was not changed after culture in CD-serum, whereas the level of hBD2-RNA expression could be induced by incubation in this medium (maximal 189%). B) The expression of hBD2, but not hBD1 is induced in inflammed gastrointestinal tissues from Crohn's patients. Conclusion: The expression of human beta defensins is differentially regulated. The expression might be influenced in vivo by circulating substances i.e. circulating cytokines that have ben demonstrated to induce I~defensin upregulation in vitro.

Background: Medullary thyroid carcinoma(MTC)arises from malignant transformation of calcitonin-secreting neuroendocrine C-cells.Pentagastrin-induced stimulation of calcitonin-release is used as a diagnostic tool in the clinical management of patients with MTC.It has previously been shown that human MTCs express the COK~-rsceptor (CCK2R).However, it has remained unclear whether CCK2R expression is restricted to MTC or whether physiological expression of this receptor occurs in normal thyroid tissue.The aim of our study was to investigate expression of the CCK2R in samples of MTC, follicular thyroid carcinoma (FTC), normal thyroid tissue and C-cell hyperplasia.Methods: 19 MTC samples, TT-cells (a human MTC cell line), three samples of normal thyroid tissue, and one FTC were analyzed for CCK2R-expression by RT-PCR.Immunohistochemistry using CCK2R-and calcitonin-specific antibodies was performed in five MTCs and one sample of non-malignant C-cell hyperplasia.Results: CCK2R expression was found in 11 of the 19 MTC samples as well as in l-r-cells.Consistent with our previous findings, the incidence of the CCK2R was high in small tumors whereas in large metastasized tumors no receptor expression was detectable.The results of pentagastrinstimulation tests correlated positively with the expression of the CCK2R.In addition to MTCs, CCK2R-expression was detected by RT-PCR in FTC as well as in all samples of normal thyroid tissue.To further localize CCK2R-expression we performed immunhistochemistry in MTC and C-cell hyperplasia samples.Staining of consecutive tissue sections with specific antibodies against the CCK2R and calcitonin revealed that normal as well as malignant C-cells express the CCK~R.Discussion: Our study shows for the first time that the CCK2R is expressed in normal thyroid tissue, specifically in C-cells.This finding suggests that in C-cells the CCK2R has a physiologic function, possibly related to bone and calcium metabolism.The rise in calcitonin serum-levels after pentagastrin-stimulation in MTCs most likely reflects an increase in C-cell number rather than a mechanism specific for malignant C-cells.Given its well established effects on growth and differentiation of ELL-ceils, gastrin might exert similar functions on the closely related C-call and in early stages of MTC.Ongoing malignant growth and potential loss of differentiation in MTC appears to be associated with a loss of CCK2R expression in advanced tumor stages.

Recombinant adenoviruses are highly efficient gene transfer vehicles but their administration to mammalians is accompanied by strong inflammatory response.The present study reports additional side effects observed during adenoviral gene transfer studies in rabbits.Hematological and serological parameters.the course of viremia and the organ distribution were analyzed after in vivo administration of EI-deleted adenoviruses in rabbits.In addition.we tested hemagglutination patterns of crude and purified virus preparations of four different E I-deleted recombinant serotype 5 adenoviruses and that of wildtype with human, rat, mice.and rabbit derived erythrocyte preparations.The systemic administration of a therapeutic dose of 5x I0" infectiou s particles/kg (infusion time 20 min) led to an average reduction of 80-90% in the platelet count within 48 h.Full recovery took 10-14 days.Virus administration induced a strong but transient erythroblastosis (peaking 24 h after administration) which settled 48 h later.Normochromic anemia occured over the next 10 days with hemoglobin levels dropping by about 40 % to reach the lowest level 10 days after administration and taking two months for full recovery.In order to test the species specific reaction, we also evaluated mice.Dose-dependent thrombocytopenia was also found in mice, but neither erythroblasto sis nor anemia was observed (in equivalent doses).The hematological findings did not improve after local injection via the portal vein.Local and systemic administration led to a comparable course of viremia .Only minor differences were found in the biodistribut ion of viruses between local and systemic administration.Large amounts of viral DNA and transgene expression were found in the lungs, the kidneys and the ovaries.even after local administration via the portal vein.Local intravenous injection via the portal vein does not prevent systemic spread of viral vectors and the occurence of vector-related side effects.We also tested the hemagglutination properties of recombinant serotype 5 adenoviruses.None of the purified serotype 5 viruses showed hemagglutination with rabbit or mice erythrocytes but partial hemagglutination in rat erythrocytes.The hematological changes in the presence of adenoviruses suggest the need for carefu l monitoring of hematological and rheological parameters in clinical trials.5171

Two types of receptors for gastrin and cholecystokinin (CCK) have been identified in the gastrointestinal tract and in the central nervous system: CCKA and CCKB receptors. Here we report evidence for the expression of CCKB receptors in the guinea-pig kidney. Specific binding sites for [125I]gastrin were detected in sections of the guinea-pig kidney: Binding was saturable, pH-, temperature- and time-dependent, and specific for gastrin-related peptides. The potencies for inhibition of binding of [125I]gastrin were CCK-8 > gastrin 17-I > CCKB receptor antagonist L-365,260 > des(SO3)CCK-8 > CCKA receptor antagonist L-364,718. Autoradiography demonstrated specific [125I]gastrin binding to medullary collecting ducts and to a much lesser extent to glomeruli, but not over other structures. CCKB receptor cDNA fragments were amplified by RT-PCR from total kidney, isolated tubuli and from tissues known to express CCKB receptors such as stomach and brain. The kidney might therefore be a previously unidentified site of action for gastrin and cholecystokinin-related peptides.