Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients. We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors. We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage. We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors.

In this study, metastasizing serotonin-producing ileocaecal carcinoid tumours (MSPCs) were examined for biological characteristics that could be used to define clinically relevant subgroups within this patient population. Possible targets for new treatment options were also explored. It was found that MSPCs share several biological characteristics such as expression of serotonin, tachykinins (TKs), chromogranin A, islet autoantigen-2 and connective tissue growth factor (CTGF). TKs and serotonin were demonstrated in the same endocrine tumours in the gut and lung. IA-2 expression was shown to be up-regulated in MSPCs, possibly in connection with active hormone secretion. CTGF expression was high in tumour areas adjacent to extensive stroma expressing alpha-smooth muscle actin. This indicated myofibroblast differentiation, which may be associated with fibrosis-related complications prevalent in patients with MSPCs. When compared with other endocrine tumours, MSPCs behaved as a relatively homogeneous group, though within the MSPC population several subgroups could be defined. Patients with tumours displaying either a solid growth pattern and/or a Ki67 index ≥1% had a less favourable prognosis than those who did not. Another group of patients, who had increased plasma TK concentrations, were more likely to suffer from severe diarrhea. This information should be considered when discussing clinical treatment and when undertaking tumour biological studies. New treatment possibilities, such as drugs that specifically target TK receptors and antibodies to CTGF, are also discussed. In conclusion, MSPCs comprise a clinically relevant tumour group with similar biological features that are distinct from other endocrine tumours. Subgroups of patients within this patient category can be defined which may be relevant when establishing prognosis and when selecting future treatment modalities.

Classical midgut carcinoids belong to neuroendocrine tumors of the gastroenteropancreatic tract (GEP-NETs) and are associated with serotonin overproduction. The term midgut is derived from the tumors’ embryological site of origin: enterochromaffin cells in the lower jejunum, ileum, caecum and the ascending colon. Despite their rather benign nature, these tumors can metastasize to mesentery and liver, putting patients at risk for the so-called carcinoid syndrome. This syndrome is characterized by flushes, diarrhoea and valvular heart disease due to the excessive serotonin secretion by tumor cells. Treatment of metastatic disease is currently ineffective and T cell immunotherapy has been suggested as a novel approach. We propose a number of midgut carcinoid-associated proteins as potential antigens for immunotherapy. Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), islet autoantigen 2 (IA-2) and survivin represent interesting candidates based on their fairly restricted neuroendocrine tissue expression. In pursuit of potential antigens we identified a novel splicing variant of VMAT-1, lacking the second last exon. The variant, denoted VMAT1Δ15, encodes a differently translated C-terminal compared to the native form, is localized in the endoplasmic reticulum (ER) instead of large dense core vesicles and is unable to accumulate serotonin. We identify several immunogenic HLA-A*0201-binding peptide epitopes derived from our proposed antigens by analyzing CD8+ T cell responses in blood from midgut carcinoid patients. We demonstrate immune recognition of midgut carcinoid tumors in patients and in vitro generation of activated CD8+ T cells recognizing these peptide epitopes in blood from healthy controls. Patients also exhibit increased frequencies of circulating regulatory T cells (Tregs) with suppressive quality and patient lymphocytes display a decreased proliferative capacity compared to healthy controls. Midgut carcinoid tumors are frequently infiltrated by T cells, however always in the presence of Foxp3-expressing Tregs. Midgut carcinoid-associated antigens recognized by CD8+ T cells are of great interest for cellular therapies such as modified DC vaccines or adoptive T cell transfer. However, the systemic and local suppression of Th1 immunity must be considered and likely corrected in order to obtain clinically effective immunotherapies.

Made available in DSpace on 2015-03-26T13:30:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4318879 bytes, checksum: f450e68114fd05e0f6ffba1575a94555 (MD5) Previous issue date: 2007-10-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The subfamily Asopinae presents a large number of predatory species, including Podisus distinctus (Stal) (Heteroptera: Pentatomidae) as one of the main species of this genus. These insects can be reared in laboratory and it presents potential for biological control of Lepidoptera defoliators in Brazilian eucalyptus plantations. This study aimed to describe the egg development (postdeposition dynamic), the immature stages (eggs and nymphs) and the midgut and salivary glands morphology of P. distinctus. This species presented eggs with oval to subglobe form, base narrower than the apex; convex circular operculum; 1.09 ± 0,03mm height; 0.90 ± 0.04mm diameter; clavated aeromicropilar processes in a circular row in the egg anterior pole, in number of 16.00 ± 2.00. The eggs of P. distinctus are similar to other species of the genus Podisus, but aspects as color, form, number of aero-micropilar processes, size and diameter can be used to differentiate them. Postdeposition alterations were observed on eggs of P. distinctus after one hour of being laid such as the change on orientation of the spines and aero-micropilar processes. The instars of P. distinctus can be identified based on characteristics presented by them, as number, arrangement and disposition of the abdominal stains and also, morphological characteristics, as total length (measured in the body medium line). Morphometric parameters as the width of the head at the eyes level and the pronotum length can also be used to identify the nymph stadiums. The recognition and differentiation of Asopinae species are easier after the third stadium when form and dorsolateral stains are more distinctive. However, the color is not a reliable character for this. Salivary glands complex and the midgut of P. distinctus are similar to those of other Asopinae species. The salivary system of P. distinctus is formed by a pair of principal bilobades glands and a pair of long and tubular accessory glands. The last one arise from the terminal portion of the accessory duct that suffers a camber. The P. distinctus principal salivary glands presents spherical and smaller anterior lobe than the posterior lobe, which has a prolonged sack form and dilated proximal portion. The P. distinctus midgut wall is formed by a simple layer of columnar cells externally covered by a well developed muscle layer, organized in a tunic composed by overlaid longitudinal and circular muscles. Podisus distinctus also present a bundle of longitudinal muscles along the whole midgut extension. / A subfamília Asopinae apresenta grande número de espécies predadoras, como Podisus distinctus (Stal) (Heteroptera: Pentatomidae), que se destaca como uma das principais desse gênero. Estes podem ser criados em laboratório e apresentam potencial para o controle biológico de lepidópteros desfolhadores em plantações de eucalipto no Brasil. Esse estudo objetivou descrever o desenvolvimento dos ovos (aspectos de formação e dinâmica pós- deposicional), os estágios imaturos (ovos e ninfas) e a morfologia do intestino médio e das glândulas salivares de P. distinctus. Essa espécie apresentou ovos com forma subglobosa a oval, base mais estreita que o ápice; opérculo circular convexo; altura de 1,09 ± 0,03mm; diâmetro de 0,90 ± 0,04mm; processos aero-micropilares clavados, em fileira circular no pólo anterior do ovo, em número de 16,00 ± 2,00. Os ovos de P. distinctus são semelhantes aos de outras espécies do gênero Podisus, mas aspectos como a cor, forma, número de processos aero-micropilares, tamanho e diâmetro dos mesmos, podem ser utilizados para diferenciá-las. Foram observadas alterações pós-deposicionais nos ovos de P. distinctus após uma hora de postura, como a mudança de orientação dos espinhos e dos processos aero-micropilares. Os estádios de P. distinctus podem ser identificados contrastando características apresentadas entre os mesmos, como o número, arranjo e a disposição das manchas abdominais dorsais e também, avaliando características morfométricas, como o comprimento total, medido na linha mediana do corpo. Parâmetros morfométricos como a largura da cabeça ao nível dos olhos e o comprimento do pronoto, podem ser utilizados em auxílio na identificação dos estádios. O reconhecimento e a diferenciação de espécies de Asopinae são facilitados a partir do terceiro estádio, quando forma e manchas dorso-laterais são características. Entretanto, a coloração não é um caracter confiável. O complexo de glândulas salivares e o intestino médio de P. distinctus são semelhantes aos de outros Asopinae. O sistema salivar de P. distinctus é formado por um par de glândulas salivares principais bilobadas e por um par de glândulas acessórias longas e tubulares. As glândulas acessórias de P. distinctus originam-se na porção terminal do ducto acessório, que sofre um abaulamento. As glândulas salivares principais de P. distinctus possuem o lóbulo anterior esférico, menor que o lóbulo posterior, que apresenta forma de saco alongado e porção proximal dilatada. A parede do intestino médio de P. distinctus é formada por uma camada simples de células colunares, revestida externamente por uma camada muscular bem desenvolvida, organizada em uma túnica externa composta por músculo longitudinal e outra interna, de músculo circular. Podisus distinctus apresentou ainda, um feixe de músculos longitudinais justapostos que segue por toda a extensão do intestino médio.

Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.

Hintergrund: Karzinoid-Tumoren des embryonalen Mitteldarms sind seltene intestinale neuroendokrine Tumoren, bei denen zum Zeitpunkt der Diagnose häufig Metastasen vorliegen. Im Gegensatz zu Karzinoiden des Vorderdarms und Respirationstraktes sind sie nicht mit der Multiplen Endokrinen Neoplasie Typ 1 (MEN1) vergesellschaftet. Die Mechanismen ihrer Tumorigenesis sind weitgehend unbekannt. Methoden: Tumorgewebe acht sporadischer, maligner Dünndarm-Karzinoide war Objekt dieser Studie über Verlust der Heterozygotie ("Loss Of Heterozygosity" (LOH)) mit 131 fluoreszierenden Mikrosatelliten. DNA Sequenz-Analyse mit Oligonucleotid Primern, die Exon 8-11 des SMAD4/DPC4 Gens flankieren sowie immunhistochemische Färbung mit Smad4/DPC4 antikörpern wurde durchgeführt. Ergebnis: Chromosom 18 wies Deletionen in 88% der Tumoren auf. Alle außer einem Tumor hatten sowohl 18p als auch 18q verloren, in einem der Tumoren war eine kleine Region telomer zu den SMAD4/DPC4/DCC Genen auf 18q21 verloren. Andere Chromosomen waren nur in drei Tumoren betroffen. LOH auf Chromosom 11q13, dem MEN1 Lokus, wurde nicht gefunden.Sequenzierung der DNA und immunhistochemische Färbung für das SMAD4/DPC4 Gen zeigten keine Aberrationen. Diskussion: Die Funde der Chromosom 18 Deletionen weisen eindeutig auf ein entscheidendes Ereignis in der Tumorigenese von Karzinoiden des Mitteldarms hin. An der Entstehung dieser Tumoren könnte ein mutmaßliches Tumor Suppressor Gen beteiligt sein, welches auf Chromosom 18 lokalisiert ist. Dahingegen ist SMAD4/DPC4 wahrscheinlich nicht in die Tumorneogenese von Carcinois Tumoren involviert. / Background: Midgut carcinoid tumors are rare malignant tumors with origin in the neuroendocrine cells of the small intestine. Due to secretion of a variety of peptide hormones and biogenic amines they cause the carcinoid syndrome. Metastases are often present at first diagnosis. Despite this, patients have a realistic chance to survive for a prolonged period (30% (unresectable/metastatic disease) -79% (non-metastatic disease) 5-year survival rate) if treated by a combination of surgery and medication. Unlike their foregut counterparts, midgut carcinoid tumors are not or rarely associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome. The genetic back-ground to tumorigenesis of these neoplasms is unknown. In contrast, the events involved in tumorigenesis of gastroenteropancreatic adenocarcinomas are better characterized with frequent mutations e.g. of the Smad4/DPC4, Smad2/MADR2/JV18-1 and DCC genes on chromosome 18. Methods: Eight metastatic midgut carcinoids were analysed by a genome-wide screening for loss of heterozygosity using 131 PCR-amplified fluorescent-labelled microsatellite markers. DNA sequence analysis using oligonucleotide primers flanking exons 8-11 of the Smad4/DPC4 gene and immunohistochemical staining with Smad4/DPC4 antibodies was performed. Results: Chromosome 18 was deleted in seven out of eight tumors (88%). All but one of these tumors had lost both 18p and 18q, the remaining tumor had lost the long arm but retained the short arm. Several other chromosomal alleles were lost in a subset of the tumors. Loss of heterozygosity (LOH) on chromosome 11q13, the MEN 1 locus, was not found. Smad4/DPC4 wild-type sequence and normal immunohistochemical staining for Smad4/DPC4 protein was found for all analysed tumors. Conclusions: Our finding of a high frequency of chromosome 18 deletions in 88% of the tumors strongly suggests that midgut carcinoid tumorigenesis might involve inactivation of a candidate tumor suppressor gene located in that region while Smad4/DPC4 is unlikely to be involved in that process. A more detailed analysis of the genetic events in midgut carcinoid tumors is warranted to clarify their neogenetic origin.

No description available.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).