Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

Complex insertions and deletions (indels) from next-generation sequencing (NGS) data were prone to escape detection by currently available variant callers as shown by large-scale human genomics studies. Somatic and germline complex indels in key disease driver genes could be missed in NGS-based genomics studies.

INDELseek is an open-source complex indel caller designed for NGS data of random fragments and PCR amplicons. The key differentiating factor of INDELseek is that each NGS read alignment was examined as a whole instead of "pileup" of each reference position across multiple alignments. In benchmarking against the reference material NA12878 genome (n = 160 derived from high-confidence variant calls), GATK, SAMtools and INDELseek showed complex indel detection sensitivities of 0%, 0% and 100%, respectively. INDELseek also detected all known germline (BRCA1 and BRCA2) and somatic (CALR and JAK2) complex indels in human clinical samples (n = 8). Further experiments validated all 10 detected KIT complex indels in a discovery cohort of clinical samples. In silico semi-simulation showed sensitivities of 93.7-96.2% based on 8671 unique complex indels in >5000 genes from dbSNP and COSMIC. We also demonstrated the importance of complex indel detection in accurately annotating BRCA1, BRCA2 and TP53 mutations with gained or rescued protein-truncating effects.

INDELseek is an accurate and versatile tool for complex indel detection in NGS data. It complements other variant callers in NGS-based genomics studies targeting a wide spectrum of genetic variations.

The present study aimed to investigate the key genes and microRNAs (miRNA/miRs) associated with coronary artery disease (CAD) progression. The gene expression profile of GSE20680 and GSE12288, and the miRNA expression profile of GSE28858 were downloaded from the gene expression omnibus database. The differentially expressed genes (DEGs) in GSE20680 and GSE12288, and the differentially expressed miRNAs in GSE28858 were screened using the limma package in R software. Common DEGs between GSE20680 and GSE12288 were selected. Functions and pathways of DEGs and miRNAs were enriched using the DAVID tool from the GO and KEGG databases. The regulatory network of miRNA and selected CAD‑associated DEGs was constructed. A total of 270 DEGs (167 upregulated and 103 downregulated) based on the GSE20680 dataset, and 2,268 DEGs (534 upregulated and 1,734 downregulated) based on the GSE12288 dataset, were screened. For the differentially expressed miRNAs, 214 were identified (102 upregulated and 112 downregulated) in CAD samples and were screened. Interferon regulatory factor 2 (IRF2) and cell death‑inducing DFFA‑like effector b (CIDEB), which are regulated by signal transducer and activator of transcription 3 and myc‑associated factor X, were identified as common DEGs for CAD. miR‑455‑5p, miR‑455‑3p and miR‑1257, which are involved in the major histocompatibility complex (MHC)protein assembly pathway and peptide antigen assembly with MHC class I protein complex pathway, may regulate various miRNAs and target genes, including pro‑opiomelancortin (POMC), toll‑like receptor 4 (TLR4), interleukin 10 (IL10), activating transcription factor 6 (ATF6) and calreticulin (CALR). The current study identified IRF2 and CIDEB as crucial genes, and miRNA‑455‑5p, miRNA‑455‑3p and miR‑1257 along with their target genes POMC, TLR4 and CALR, as miRNAs involved in CAD progression. Thus, the present study may provide a basis for future research into the progression mechanism of CAD.

BACKGROUND

True essential thrombocythemia (ET) may carry one of the known driver mutations (JAK2, MPL and CALR) or none of them [in triple-negative (3NEG) cases]. The patients' mutational status seems to delineate the clinical manifestations of ET.

METHODS

We report the data of 183 patients diagnosed with ET strictly according to the WHO 2008 criteria and with a full molecular diagnosis, including the following: 114 patients (62·3%) with JAK2V617F; 25 (13·7%) with CALR type 1 and 19 (10·4%) with CALR type 2; 3 (1·6%) with MPL; 22 (12%) who were 3NEG. Thrombotic risk was assessed by means of the IPSET-thrombosis score (IPSET-T).

RESULTS

CALR and 3NEG patients had lower haemoglobin levels and leucocyte count than JAK2 patients. CALR patients, and those with type 2 in particular, had higher mean platelet counts and had extreme thrombocytosis more often than any of the other groups. Based on their IPSET-T stratification, 3NEG- and CALR-mutated patients belonged more frequently to the low-risk group and had a significant more favourable thrombosis-free survival rate than those with JAK2 mutation.

CONCLUSIONS

These findings indicate that the three different molecular markers have a significant impact on the clinical course of true ET, giving rise to different phenotypes of the same disease.

Currently, prognostication in primary myelofibrosis (PMF) relies on the International Prognostic Scoring System (IPSS), dynamic IPSS (DIPSS), and DIPSS-plus, which incorporate age, blood counts, constitutional symptoms, circulating blasts, red cell transfusion need, and karyotype. Although the JAK2 V617F mutation was discovered a decade ago and MPL mutations shortly thereafter, it was the recent discovery of CALR mutations in the vast majority of JAK2/MPL-unmutated patients and recognition of the powerful impact of CALR mutations and triple-negative (JAK2/MPL/CALR-negative) status on outcome that set the stage for revision of traditional prognostic models to include molecular information. Additionally, the advent of next-generation sequencing has identified a host of previously unrecognized somatic mutations across hematologic malignancies. As in the myelodysplastic syndromes, the majority of common and prognostically informative mutations in PMF affect epigenetic regulation and mRNA splicing. Thus, a need has arisen to incorporate mutational information on genes such as ASXL1 and SRSF2 into risk stratification systems. Mutations in yet other genes appear to be important players in leukemic transformation, and new insights into disease pathogenesis are emerging. Finally, the number of prognostically detrimental mutations may affect both survival and response to ruxolitinib, which has significant implications for clinical decision making. In this review, we briefly summarize the prognostic models in use today and discuss in detail the somatic mutations commonly encountered in patients with PMF, along with their prognostic implications and role in leukemic transformation. Emerging prognostic models that incorporate new molecular information into existing systems or exclude clinical variables are also presented.

The protective effect of hydrogen sulfide (H2S) against hypoxia-induced injury via anti-apoptosis is well established, but the underlying mechanism remains unclear. The present study aimed to investigate whether miR-455 participated in the H2S protection of lung epithelial cells against CoCl2-induced apoptosis by regulating endoplasmic reticulum stress (ERS)-related genes. Human lung epithelial cells BEAS-2B were subjected to hypoxia injury with or without H2S preconditioning. It was found that hypoxia injury increased apoptosis of BEAS-2B cells, down-regulated the expression of miR-455, and upregulated the expression of calreticulin (Calr). H2S preconditioning attenuated lung epithelial cells apoptosis, enhanced cell viability, up-regulated the expression of miR-455, as well as down-regulated the expression of Calr following hypoxia injury. In addition, Calr, GRP78, C/EBP homologous protein (CHOP) and Caspase-12 protein was down-regulated by the miR-455 mimic and up-regulated by the miR-455 inhibitor. These results implicate miR-455 regulated H2S protection of lung epithelial cells against hypoxia-induced apoptosis by stimulating Calr.

Objective: Emerging evidence suggests that calreticulin (CALR) has great impacts on the tumor formation and progression of various cancers, but the role of CALR remains controversial. We investigated the expression and clinical significance of CALR in nasopharyngeal carcinoma (NPC). Methods: Immunohistochemistry was used to detect the expression of CALR in NPC tissues, and the correlation of CALR with clinicopathological characteristics and prognosis were analyzed. The cell functions of CALR in NPC cells were also performed in vitro. Results: Compared with non-tumor nasopharyngeal epithelium (NPE) tissues, CALR expression was markedly up-regulated in NPC tissues (P < 0.001), and the high expression of CALR was positively associated with advanced clinical stage (P=0.003) and metastasis (P=0.023). Compared to the patients with low expression of CALR, patients who displayed high expression of CALR may achieve a poorer progression-free survival (PFS) and overall survival (OS) (P < 0.001). Furthermore, multivariate analysis showed that high expression of CALR was an independent predictor of poor prognosis. In addition, we found that knockdown of CALR significantly inhibited the proliferation, migration and invasion of CNE2 and HONE1 cells in vitro, and the mechanism might be associated with inactivation of Stat3 signaling pathway. Conclusion: CALR may promote NPC progression and metastasis via involving Stat3 signaling pathway, and can be regarded as an effective potential predictor for progression and prognosis of NPC.

We introduce a 78-year-old woman presented with thrombocytosis and high blast count who had a history of splenectomy. Her cytogenetic analysis revealed aberrant chromosomal rearrangements in different clonal populations harboring 46XX karyotype with t(9;22) (q34;q11). RT-PCR assay detected the e1a2 BCR-ABL translocation resulting from rearrangement of the minor breakpoint cluster region (m-bcr) in BCR gene. Subsequent evaluation of the disease showed calreticulin (CALR) 52-bp deletion as well as the absence of JAK2 (V617F) heterozygous mutation in granulocyte population of peripheral blood using allele-specific PCR and bi-directional DNA sequencing. To our knowledge, this is the first case of a patient initially diagnosed as p190 BCR-ABL transcript positive CML in blast crisis characterized by a 52-bp deletion in CALR gene.

BACKGROUND

Primary myelofibrosis (PMF) is quite rare in children. Mutations of JAK2(V617F) or MPL(W515K/L) were absent in pediatric patients with PMF according to previous studies. Recently, mutations in calreticulin (CALR) were described in adult patients with JAK2/MPL-unmutated PMF. Our study aimed to analyze the clinical and genetic features of Chinese pediatric patients with PMF.

METHODS

We retrospectively investigated 14 pediatric patients diagnosed as PMF according to WHO 2008 criteria. Direct sequencing was performed for the existence of genetic alterations in JAK2, MPL, TET2, CBL, ASXL1, IDH1, IDH2, SRSF2, EZH2, DNMT3A and CALR.

RESULTS

In our cohort, all patients had anemia, three patients (21%) had splenomegaly, six patients (43%) had micromegakaryocytes at time of diagnosis. No patient had spontaneous remission and six patients (43%) transformed to acute myelocytic leukemia. In nine patients with evaluable cytogenetic information, three subjects (33%) had abnormal karyotypes. The median survival from time of diagnosis was 28 months. Seven patients (50%) had type 2 mutations of CALR. No patient had mutations in the other candidate genes. There was no statistical differences in age, gender, hemoglobin, WBC, neutrophil and platelet counts, percentage of circulating blast, overall survival and leukemia transformation between patients with and without CALR mutation.

CONCLUSIONS

Our study documented that Chinese pediatric patients with PMF in our cohort had its own clinical characteristics and poor outcome. CALR mutations were detected in 50% of our pediatric patients with PMF. Based on our study, CALR mutations screening could be used as molecular marker for diagnosis of pediatric patients with PMF.

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2 years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2-0·9, P = 0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9-4·6, P = 0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1-5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1-3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343 days vs. 95 days, P = 0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival.

Connections from intrinsic primary afferent neurons (IPANs), to ascending motor and interneurons have been described in guinea pig colon. These mono- and polysynaptic circuits may underlie polarized motor reflexes evoked by local gut stimulation. There is a need to translate findings in guinea pig to mouse, a species increasingly used in enteric neuroscience. Here, mouse distal colon was immunolabelled for CGRP, a marker of putative IPANs. This revealed a combination of large, intensely immunofluorescent axons in myenteric plexus and circular muscle, and thinner varicose axons with less immunofluorescence. The latter formed dense, basket-like varicosity clusters (CGRP+ baskets) that enveloped myenteric nerve cell bodies. Immunolabelling after 4-5 days in organ culture caused loss of smooth CGRP+ axons, but not varicose CGRP+ fibres and CGRP+ baskets. Baskets were characterized further by triple labelling with CGRP, nitric oxide synthase (NOS) and calretinin (CALR) antibodies. Approximately half (48%) of nerve cell bodies inside CGRP+ baskets lacked both NOS and CALR, while 2 overlapping populations containing NOS and/or CALR comprised the remainder. Quantitative analysis revealed CGRP+ varicosities were most abundant in baskets, followed by CALR+ varicosities, with a high degree of co-localisation between the two markers. Few NOS+ varicosities occurred in baskets. Significantly higher proportions of CALR+ and CGRP+ varicosities colocalized in baskets than in circular muscle. In conclusion, CGRP+ baskets in mouse colon are formed by intrinsic enteric neurons with a neurochemical profile consistent with IPANs and have direct connections to both excitatory and inhibitory neurons. This article is protected by copyright. All rights reserved.

Myeloproliferative neoplasms (MPN) are clonal hematological malignancies that lead to overproduction of mature myeloid cells. They are due to acquired mutations in genes encoding for AK2, MPL and CALR that result in the activation of the cytokine receptor/JAK2 signaling pathway. In addition, it exists germline variants that can favor the initiation of the disease or may affect its phenotype. First, they can be common risk alleles, which correspond to frequent single nucleotide variants present in control population and that contribute to the development of either sporadic or familial MPN. Second, some variants predispose to the onset of MPN with a higher penetrance and lead to familial clustering of MPN. Finally, some extremely rare genetic variants can induce MPN-like hereditary disease. We will review these different subtypes of germline genetic variants and discuss how they impact the initiation and/or development of the MPN disease.

Keywords: CNV; Germline variants; Risk allele; SNV; Sporadic and familial MPN.

Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by an increased risk of thrombosis and progression to acute myeloid leukemia. MPN are associated with driver mutations in JAK2, CALR and MPL which are crucial for the diagnosis and lead to a constitutive activation of the JAK-STAT signaling, independent of cytokine regulation. Moreover, most patients have concomitant mutations in genes involved in DNA methylation, chromatin modification, messenger RNA splicing, transcription regulation and signal transduction. These additional mutations may arise before, in the context of clonal hematopoiesis of indeterminate potential (CHIP), or after the acquisition of the driver mutation. The clinical phenotype of MPN results from complex interactions between mutations and host factors. The increased application of next-generation sequencing (NGS) techniques to a large series of patients with MPN has expanded the knowledge of mutational landscape and contributed to define the clinical significance of mutations. This molecular information is being increasingly used to refine diagnosis, risk stratification, monitoring of residual disease and response to treatment. ASXL1, SRSF2, EZH2, IDH1/IDH2 and U2AF1 mutations are associated with a more advanced disease and reduced overall survival in primary myelofibrosis (PMF), whereas spliceosome mutations in Polycythemia vera (PV) and essential thrombocythemia (ET) adversely affect both overall (SF3B1, SRSF2 in ET and SRSF2 in PV) and myelofibrosis-free (U2AF1, SF3B1 in ET) survival. This review discusses current knowledge of the molecular landscape of MPN, and how the availability of those molecular information may impact patient management.

Keywords: JAK-STAT pathway; essential thrombocythemia; gene mutations; myelofibrosis; myeloproliferative neoplasms; polycythemia vera.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms characterized by increased rate of cardiovascular events, a varying burden of symptoms, and an intrinsic risk of evolution to secondary forms of myelofibrosis and acute leukemia; however, survival is only modestly reduced in most instances. In the last few years, following the description of driver mutations in JAK2, MPL and CALR, the diagnostic criteria for PV and ET were revised, making the identification of very early stages feasible. Scores for identifying patients at different risk of thrombosis were refined, and they largely guide therapeutic decisions. Treatment is therefore mainly focused on reduction of thrombosis risk, control of myeloproliferation, improvement of symptomatic burden, and management of disease-associated complications. New drugs recently entered the clinical arena, with the promise to improve overall patients' management. However, evidence of a disease-modifying potential is largely missing and represents a still unmet clinical need.

Keywords: Essential thrombocythemia; Hydroxyurea; JAK2; Polycythemia vera; Thrombosis.

Calreticulin (CALR) mutations have recently been reported in 70-84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of <4% and a significantly higher platelet count compared with JAK2 V617F (P < 0.001) and CALR (P = 0.001) single-mutation patients. The findings from this study support the possibility of coexisting mutations of the JAK2, CALR, and MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications.

Neoadjuvant chemoradiotherapy (nCRT) following surgery significantly improves the survival rate of patients with rectal cancer. However, nCRT is associated with significant adverse symptoms and high medical costs. Therefore, it is important to investigate potential biomarkers for the prediction of the response to nCRT in patients with rectal cancer. The present study identified candidate biomarkers for predicting a complete response (CR) to nCRT in patients with rectal cancer and investigated the associated mechanisms. Microarray data (accession no. GSE29298) was downloaded from the Gene Expression Omnibus database. Differentially expressed microRNAs (miRNAs/miR) were screened between the pathological CR (pCR) group and no pCR (incomplete response) group. miRNA target genes were predicted using the miRWalk 2.0 online tool and subjected to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Furthermore, a miRNA co‑regulatory network was constructed and disease‑associated genes were predicted. The results demonstrated that a total of 36 upregulated and 5 downregulated miRNAs were identified between the two groups. Among these differentially expressed miRNAs, miR‑548c‑5p, miR‑548d‑5p and miR‑663a were significantly associated with a CR to nCRT. The co‑regulatory network and pathway analysis indicated that miR‑548c‑5p and miR‑548d‑5p may function together through stem cell pluripotency and ubiquitin‑mediated proteolysis signaling pathways. Furthermore, the prediction of disease‑associated genes demonstrated that miR‑548c‑5p/miR‑548d‑5p and miR‑663a may regulate genes associated with rectal cancer, including mutated in colorectal cancers (MCC) and adenomatous polyposis coli (APC), and colorectal neoplasms, including interleukin‑6 signal transducer (IL6ST), cell cycle checkpoint kinase 2 (CHEK2), marker of proliferation Ki‑67 (MKI67), cadherin 7 (CDH7), calreticulin (CALR) and transforming growth factor β1 (TGFB1). Therefore, miR‑548c‑5p, miR‑548d‑5p and miR‑663a are promising candidate biomarkers for predicting a CR to nCRT. miR‑548c‑5p/miR‑548d‑5p may be associated with a CR by regulating IL6ST, CHEK2, MKI67 and MCC. In addition, it may function through the pluripotency of stem cells and ubiquitin‑mediated proteolysis signaling pathways. miR‑663a may be associated with a CR to nCRT by targeting CDH7, CALR, APC and TGFβ1. Thus, the miRNA biomarkers investigated in the present study may represent novel therapeutic targets for the prediction and eventual improvement of the response to nCRT in patients with rectal cancer.

The 2016 revised WHO criteria for the diagnosis of pre-fibrotic/early primary myelofibrosis (pre-PMF) require at least one of the following four borderline expressed minor clinical criteria: anemia, leukocytosis, elevated lactate dehydrogenase and splenomegaly. In this study, we evaluated the relative frequency of these four criteria in a group of 170 pre-PMF patients and compared them to 225 ET cases. More than 91% of pre-PMF cases showed one or more of these features required for diagnosis, by contrast with only 48% of ET patients. According to clinical data the cumulative risk of progression to advanced/overt PMF in pre-PMF was 36.9% after 15 years. After fitting cox regression models to analyze the impact of the minor criteria on overall survival, only leukocytosis remained as a significant predictor of survival in both pre-PMF and ET. Molecular characterization showed differences in survival in pre-PMF but not ET, with CALR being a more favorable mutation than JAK2. The different outcome of pre-PMF versus ET and associated molecular genetic data supports the concept of two different entities, rather than a continuum of the same disease. Although slightly less than 50% of ET patients also show one or more minor clinical criteria, accurate distinction between ET and pre-PMF is possible by following an integrated approach including histomorphological diagnosis and presence of minor clinical criteria.

OBJECTIVE

To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice.

METHODS

The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method.

RESULTS

The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm(2)) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG.

CONCLUSIONS

Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons.

We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.