Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host.

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

An Alba-domain protein required for proteome remodelling during trypanosome differentiation and host transition.

The transition between hosts is a challenge for digenetic parasites as it is unpredictable. For Trypanosoma brucei subspecies, which are disseminated by tsetse flies, adaptation to the new host requires differentiation of stumpy forms picked up from mammals to procyclic forms in the fly midgut. Here we show that the Alba-domain protein Alba3 is not essential for mammalian slender forms, nor is it required for differentiation of slender to stumpy forms in culture or in mice. It is crucial, however, for the development of T. brucei procyclic forms during the host transition. While steady state levels of mRNAs in differentiating cells are barely affected by the loss of Alba3, there are major repercussions for the proteome. Mechanistically, Alba3 aids differentiation by rapidly releasing stumpy forms from translational repression and stimulating polysome formation. In its absence, parasites fail to remodel their proteome appropriately, lack components of the mitochondrial respiratory chain and show reduced infection of tsetse. Interestingly, Alba3 and the closely related Alba4 are functionally redundant in slender forms, but Alba4 cannot compensate for the lack of Alba3 during differentiation from the stumpy to the procyclic form. We postulate that Alba-domain proteins play similar roles in regulating translation in other protozoan parasites, in particular during life-cycle and host transitions.

Flagellar cAMP signaling controls trypanosome progression through host tissues.

The unicellular parasite Trypanosoma brucei is transmitted between mammals by tsetse flies. Following the discovery that flagellar phosphodiesterase PDEB1 is required for trypanosomes to move in response to signals in vitro (social motility), we investigated its role in tsetse flies. Here we show that PDEB1 knockout parasites exhibit subtle changes in movement, reminiscent of bacterial chemotaxis mutants. Infecting flies with the knockout, followed by live confocal microscopy of fluorescent parasites within dual-labelled insect tissues, shows that PDEB1 is important for traversal of the peritrophic matrix, which separates the midgut lumen from the ectoperitrophic space. Without PDEB1, parasites are trapped in the lumen and cannot progress through the cycle. This demonstrates that the peritrophic matrix is a barrier that must be actively overcome and that the parasite's flagellar cAMP signaling pathway facilitates this. Migration may depend on perception of chemotactic cues, which could stem from co-infecting parasites and/or the insect host.

Marinesco-Sjögren syndrome protein SIL1 regulates motor neuron subtype-selective ER stress in ALS.

Mechanisms underlying motor neuron subtype-selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic signatures. We identify cochaperone SIL1, mutated in Marinesco-Sjögren syndrome (MSS), as being robustly expressed in disease-resistant slow motor neurons but not in ER stress-prone fast-fatigable motor neurons. In a mouse model of MSS, we demonstrate impaired ER homeostasis in motor neurons in response to loss of SIL1 function. Loss of a single functional Sil1 allele in an ALS mouse model (SOD1-G93A) enhanced ER stress and exacerbated ALS pathology. In SOD1-G93A mice, SIL1 levels were progressively and selectively reduced in vulnerable fast-fatigable motor neurons. Mechanistically, reduction in SIL1 levels was associated with lowered excitability of fast-fatigable motor neurons, further influencing expression of specific ER chaperones. Adeno-associated virus-mediated delivery of SIL1 to familial ALS motor neurons restored ER homeostasis, delayed muscle denervation and prolonged survival.

Early over-expression of GRP receptors in prostatic carcinogenesis.

BACKGROUND: The GRP receptor shows high over-expression in prostatic adenocarcinoma and high grade PIN, but low expression in normal prostate glands. This represents the molecular basis for GRP receptor imaging of prostate cancer with radioactive compounds. However, a focal, high density GRP receptor expression can be observed in hitherto uncharacterized prostate glands. METHODS: GRP receptors were quantitatively measured with in vitro receptor autoradiography using ¹²5I-Tyr4 -bombesin in samples from 115 prostates. On successive tissue sections, ¹²5I-Tyr4 -bombesin autoradiography was compared with H&E staining and MIB-1 and 34ßE12 immunohistochemistry. RESULTS: On one hand, it was confirmed that GRP receptors were expressed in adenocarcinoma and high grade PIN in high density and high incidence (77% and 73%, respectively), but in normal prostate glands in low density and low frequency (18%). On the other hand, a novel and intriguing observation was the existence of focal non-invasive prostate glands with high GRP receptor density, characterized by low grade nuclear atypia and increased proliferation, compatible with lower grade PIN. There was a significant GRP receptor density gradient (P ≤ 0.005), increasing from normal prostate glands (mean relative optical density, ROD, of ¹²5I-Tyr4 -bombesin binding: 0.17) over atypical glands without increased MIB-1 labeling (0.28) and atypical glands with increased MIB-1 expression (0.44) to high grade PIN and adenocarcinoma (0.64 and 0.58, respectively). CONCLUSIONS: GRP receptor over-expression may be a novel, specific marker of early prostatic neoplastic transformation, arising in low grade PIN, and progressively increasing during malignant progression. This should be considered when interpreting in vivo GRP receptor imaging in males.

GLP-2 receptors in human disease: high expression in gastrointestinal stromal tumors and Crohn's disease.

Peptide hormones of the glucagon-like peptide (GLP) family play an increasing clinical role, as reported for GLP-1 in diabetes therapy and insulinoma diagnostics. GLP-2, despite its known trophic and anti-inflammatory intestinal actions translated into preliminary clinical studies using the GLP-2 analogue teduglutide for treatment of short bowel syndrome and Crohn's disease, remains poorly characterized in terms of expression of its receptor in tissues of interest. Therefore, the GLP-2 receptor expression was assessed in 237 tumor and 148 non-neoplastic tissue samples with in vitro receptor autoradiography. A GLP-2 receptor expression was present in 68% of gastrointestinal stromal tumors (GIST). Furthermore, GLP-2 receptors were identified in the intestinal myenteric plexus, with significant up-regulation in active Crohn's disease. The GLP-2 receptors in GIST may be used for clinical applications like in vivo targeting with radiolabelled GLP-2 analogues for imaging and therapy. Moreover, the over-expressed GLP-2 receptor in the myenteric plexus may represent the morphological correlate of the clinical target of teduglutide in Crohn's disease.

Glucose-dependent insulinotropic polypeptide receptors in most gastroenteropancreatic and bronchial neuroendocrine tumors.

CONTEXT: Gastrointestinal peptide hormone receptors overexpressed in neuroendocrine tumors (NET), such as somatostatin or glucagon-like peptide-1 (GLP-1) receptors, are used for in vivo tumor targeting. Unfortunately, not all NET express these receptors sufficiently. OBJECTIVE: Our aim was to evaluate in vitro the expression of another incretin receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, in human tumors and compare it with that in adjacent nonneoplastic tissues and also with somatostatin and GLP-1 receptor expression. METHODS: GIP receptor protein expression was qualitatively and quantitatively investigated in 260 human tumors and in nonneoplastic human tissues with receptor autoradiography using [(125)I]GIP(1-30). Pharmacological competition experiments and mRNA analysis were performed to provide proof of specificity. Somatostatin receptor and GLP-1 receptor autoradiography were performed in adjacent sections. RESULTS: GIP receptors are expressed in the majority of pancreatic, ileal, and bronchial NET. Importantly, most of the somatostatin receptor-negative NET and GLP-1 receptor-negative malignant insulinomas are GIP receptor positive. Conversely, the epithelial and stromal gastrointestinal tumors, including gastric, colonic, and hepatocellular carcinomas, cholangiocarcinomas, and gastrointestinal stromal tumors as well as lung adenocarcinomas are usually GIP receptor negative, except for 26% of pancreatic adenocarcinomas. Pancreatic islets, but not acini, are GIP receptor positive. The rank order of potencies for receptor binding and mRNA analysis by PCR reveal specific GIP receptors. CONCLUSIONS: The numerous GIP receptors in gastroenteropancreatic and bronchial NET represent novel universal molecular targets for clinical applications, in particular for in vivo scintigraphy and targeted radiotherapy. These results may also be the basis for multiple targeting, with concomitant use of GIP, somatostatin, and GLP-1 analogs as radiotracers.

Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: molecular basis for dual targeting of tumoral vasculature.

Gastrin-releasing peptide (GRP) and GRP receptors (GRPR) play a role in tumor angiogenesis. Recently, GRPR were found to be frequently expressed in the vasculature of a large variety of human cancers. Here, we characterize these GRPR by comparing the vascular GRPR expression and localization in a selection of human cancers with that of an established biological marker of neoangiogenesis, the vascular endothelial growth factor (VEGF) receptor. In vitro quantitative receptor autoradiography was performed in parallel for GRPR and VEGF receptors (VEGFR) in 32 human tumors of various origins, using ¹²5I-Tyr-bombesin and ¹²5I-VEGF165 as radioligands, respectively. Moreover, VEGFR-2 was evaluated immunohistochemically. All tumors expressed GRPR and VEGFR in their vascular system. VEGFR were expressed in the endothelium in the majority of the vessels. GRPR were expressed in a subpopulation of vessels, preferably in their muscular coat. The vessels expressing GRPR were all VEGFR-positive whereas the VEGFR-expressing vessels were not all GRPR-positive. GRPR expressing vessels were found immunohistochemically to co-express VEGFR-2. Remarkably, the density of vascular GRPR was much higher than that of VEGFR. The concomitant expression of GRPR with VEGFR appears to be a frequent phenomenon in many human cancers. The GRPR, localized and expressed in extremely high density in a subgroup of vessels, may function as target for antiangiogenic tumor therapy or angiodestructive targeted radiotherapy with radiolabeled bombesin analogs alone, or preferably together with VEGFR targeted therapy.

Wild-type and splice-variant secretin receptors in lung cancer: overexpression in carcinoid tumors and peritumoral lung tissue.

Gastrointestinal peptide hormone receptors, like somatostatin receptors, are often overexpressed in human cancer, allowing receptor-targeted tumor imaging and therapy. A novel candidate for these applications is the secretin receptor recently identified in pancreatic and cholangiocellular carcinomas. In the present study, secretin receptors were assessed in a non-gastrointestinal tissue, the human lung. Non-small-cell lung cancers (n=26), small-cell lung cancers (n=10), bronchopulmonary carcinoid tumors (n=29), and non-neoplastic lung (n=46) were investigated for secretin receptor protein expression with in vitro receptor autoradiography, using (125)I-[Tyr(10)] rat secretin and for secretin receptor transcripts with RT-PCR. Secretin receptor protein expression was found in 62% of bronchopulmonary carcinoids in moderate to high density, in 12% of non-small cell lung cancers in low density, but not in small cell lung cancers. In tumors found to be secretin receptor positive by autoradiography, RT-PCR revealed transcripts for the wild-type secretin receptor and for novel secretin receptor splice variants. In the non-neoplastic lung, secretin receptor protein expression was observed in low density along the alveolar septa in direct tumor vicinity in cases of acute inflammation, but not in histologically normal lung. In the autoradiographically positive peritumoral lung, RT-PCR showed transcripts for the wild-type secretin receptor and for a secretin receptor spliceoform different from those occurring in lung and gut tumors. In conclusion, secretin receptors are new markers for bronchopulmonary carcinoid tumors, and represent the molecular basis for an in vivo targeting of carcinoid tumors for diagnosis and therapy. Furthermore, secretin receptors may play a role in peritumoral lung pathophysiology. Secretin receptor mis-splicing specifically occurs in tumor and non-tumor lung pathology.

CRF receptors in the rodent and human cardiovascular systems: species differences.

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Secretin receptors in the human liver: expression in biliary tract and cholangiocarcinoma, but not in hepatocytes or hepatocellular carcinoma.

BACKGROUND/AIMS: Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer. METHODS: Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using (125)I-[Tyr(10)] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR. RESULTS: Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas. CONCLUSIONS: The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.

Secretin receptors in normal and diseased human pancreas: marked reduction of receptor binding in ductal neoplasia.

Receptors for gut hormones, which are often overexpressed in cancer, are clinically relevant for receptor-targeted tumor imaging and therapy. Because the receptors for the gut hormone secretin are poorly characterized, we assessed secretin receptor expression in the main secretin target, the human pancreas. We investigated 58 non-neoplastic pancreases and 55 pancreatic tumors for receptor localization and density by in vitro receptor autoradiography using [(125)I]Tyr(10) rat secretin and for secretin receptor mRNA by reverse transcriptase-polymerase chain reaction. Secretin receptors were highly expressed in non-neoplastic ducts and lobuli and also in lower amounts in ductal neoplasias, including ductal adenocarcinoma, intraductal papillary mucinous tumors, and pancreatic intraepithelial neoplasia. Reverse transcriptase-polymerase chain reaction revealed wild-type receptor mRNA in the non-neoplastic pancreas and both wild-type and spliced variant receptor transcripts in ductal adenocarcinomas. Serous cystic tumors were highly positive for secretin receptors, whereas mucinous cystic tumors were negative. This study is the first to describe the precise secretin receptor distribution in human non-neoplastic pancreas and various pancreatic tumors. High secretin receptor expression in the non-neoplastic ducts reflects the major role of secretin in bicarbonate secretion. Reduced secretin binding in pancreatic ductal tumors may relate to (alternatively spliced) secretin receptor isoforms. Thus, secretin receptors in pancreatic tumors may represent potential clinical targets.