Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Abnormal basement membrane results in increased keratinocyte-derived periostin expression in psoriasis similar to wound healing.

The psoriatic skin resembles wound healing, and it shows abnormalities at the basement membrane (BM), also in the non-lesional skin. Fibroblast-derived dermal periostin has well-known functions in wound healing and Th2-mediated diseases, such as atopic dermatitis. Here we show that serum periostin level was elevated in psoriatic patients, remarkably in the systemically treated ones. Obvious periostin positivity was detected in basal keratinocytes of the non-lesional, lesional, and previously-lesional psoriatic vs. healthy skin. Ex vivo skin models were generated to examine how different skin injuries affect periostin expression during wound healing. Our newly developed cultured salt-split model demonstrated that BM-injury induced periostin expression in basal keratinocytes, and periostin levels in the supernatant were also increased upon healing. In wound healing models, ß1-integrin expression was similarly induced. ß1-integrin blocking caused reduced periostin expression in in vitro scratch assay, indicating that ß1-integrin can mediate periostin production. In contrast to atopic dermatitis, psoriatic basal keratinocytes are in an activated state and show a stable wound healing-like phenotype with the overexpression of periostin. This abnormal BM-induced wound healing as a potential compensatory mechanism can be initiated already in the non-lesional skin present in the lesion and keratinocytes can remain activated in the healed skin.

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells.

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation.

The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo.

OBJECTIVE: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

Reconstruction of Alar-Perialar Defects with a Combined Subcutaneous and Cutaneous Pedicled Rotation-Advancement Nasolabial Flap.

Aim of the Study: During the reconstruction of alar defects involving the upper lip, reconstructive surgeons face the need for various thicknesses of tissues crucial to preserving the facial sulcus which is important for a cosmetically acceptable result. Our aim was to reconstruct the deep perialar and thinner lateral nasal alar defect in a single step procedure with a suitable flap which is reliable, has appropriate blood supply and provides an esthetically good result. Materials and Methods: Extended alar defect was reconstructed with a combined flap in 10 cases. During the procedure, a subcutaneous pedicle was created and the proximal part of the flap was rotated into the defect as a rotational flap. The procedure and the follow-up have been photo-documented in all cases. Furthermore, the perfusion of the flaps was monitored by means of laser Doppler flowmetry. Postoperative complications were evaluated with a semi-quantitative score and the patients completed a patient satisfaction questionnaire, too. Results: An optimal esthetic result was obtained in all cases after the operation. The lateral nasal alar part of the defect was reconstructed with the thinner proximal part of the flap while the deeper perialar region involving the upper lip was covered with the thicker distal part. The flaps have shown sufficient blood flow after the operation. There was no significant pin cushioning or "trap-door" effect in any case. Mild erythema and edema was found in few cases. The patients were satisfied with the cosmetic result of the intervention. Conclusions: The flap is suitable for the reconstruction of alar defects involving the perialar region. It has the advantage of covering the deeper perialar and the thinner alar defects, whilst eliminating the pin cushioning effect of the conventional subcutaneous island pedicle flaps.

A Possible Technique for the Complex Reconstruction of Exposed Breast Implant: Applicability and Microcirculation of the Capsule Flap.

Aim of the Study: Immediate breast reconstruction is often applied after mastectomy. However, inappropriate surgical technique, postoperative radiotherapy and infection may lead to tissue necrosis and implant protrusion. Traditional therapies frequently fail. However, previous data suggested that capsule flaps may be appropriate for the salvage of implants. Our goal was to investigate the usefulness of capsuloplasty in patients with exposed breast implant and to monitor the blood supply of capsule flaps during the operation. Materials and Methods: Capsuloplasty was performed in 19 patients with exposed implant. After removal of necrotic tissue, capsulotomy was performed, the planned flap was dissected free, the implant was covered with the flap and the wound was then closed. During operation, the blood flow of the flap was determined by means of laser Doppler flowmetry. Moreover, tissue samples were taken for histology and immunostaining for CD34. Results: The postoperative follow-up showed that capsule flaps survived in each case: no complications were found. The blood flow of the flaps did not change significantly during the intervention as compared with the baseline values. The histology and the immunohistochemistry revealed considerable vascularization and angiogenesis in the flap. Conclusions: Capsule flaps seem to be appropriate for the salvage of exposed implants and for enhancement of implant cover in the case of thin and injured tissue.

Climate-induced phenological shift of apple trees has diverse effects on pollinators, herbivores and natural enemies.

Climate change is altering the phenology of trophically linked organisms, leading to increased asynchrony between species with unknown consequences for ecosystem services. Although phenological mismatches are reported from several ecosystems, experimental evidence for altering multiple ecosystem services is hardly available. We examined how the phenological shift of apple trees affected the abundance and diversity of pollinators, generalist and specialist herbivores and predatory arthropods. We stored potted apple trees in the greenhouse or cold store in early spring before transferring them into orchards to cause mismatches and sampled arthropods on the trees repeatedly. Assemblages of pollinators on the manipulated and control trees differed markedly, but their overall abundance was similar indicating a potential insurance effect of wild bee diversity to ensure fruit set in flower-pollinator mismatch conditions. Specialized herbivores were almost absent from manipulated trees, while less-specialized ones showed diverse responses, confirming the expectation that more specialized interactions are more vulnerable to phenological mismatch. Natural enemies also responded to shifted apple tree phenology and the abundance of their prey. While arthropod abundances either declined or increased, species diversity tended to be lower on apple trees with shifted phenology. Our study indicates novel results on the role of biodiversity and specialization in plant-insect mismatch situations.

Matrix metalloproteinase (MMP)-7 in Barrett's esophagus and esophageal adenocarcinoma: expression, metabolism, and functional significance.

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.

Cell cycle dependent expression of the CCK2 receptor by gastrointestinal myofibroblasts: putative role in determining cell migration.

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract.

Glucagon-like petide-2 acts on colon cancer myofibroblasts to stimulate proliferation, migration and invasion of both myofibroblasts and cancer cells via the IGF pathway.

Glucagon-like peptide (GLP)-2 stimulates intestinal epithelial proliferation by acting, in part, via IGF release from sub-epithelial myofibroblasts. The response of myofibroblasts to GLP-2 remains incompletely understood. We studied the action of GLP-2 on myofibroblasts from colon cancer and adjacent tissue, and the effects of conditioned medium from these cells on epithelial cell proliferation, migration and invasion. GLP-2 stimulated proliferation, migration and invasion of myofibroblasts and the proliferative and invasive responses of cancer-associated myofibroblasts were greater than those of myofibroblasts from adjacent tissue. The responses were inhibited by an IGF receptor inhibitor, AG1024. Conditioned medium from GLP-2 treated myofibroblasts increased proliferation, migration and invasion of SW480, HT29, LoVo epithelial cells and these responses were inhibited by AG1024; GLP-2 alone had no effect on these cells. In addition, when myofibroblasts and epithelial cells were co-cultured in Ibidi chambers there was mutual stimulation of migration in response to GLP-2. The latter increased both IGF-1 and IGF-2 transcript abundance in myofibroblasts. Moreover, a number of IGF binding proteins (IGFBP-4, -5, -7) were identified in myofibroblast medium; in the presence of GLP-2 there was increased abundance of the cleavage products of IGBBP-4 and IGFBP-5 suggesting activation of a degradation mechanism that might increase IGF bioavailability. The data suggest that GLP-2 stimulates cancer myofibroblast proliferation, migration and invasion; GLP-2 acts indirectly on epithelial cells partly via increased IGF expression in myofibroblasts and partly, perhaps, by increased bioavailability through degradation of IGFBPs.

[Comparison of surgical patients with gastroesophageal reflux disease and Barrett's esophagus]. / Refluxbetegség és Barrett-nyelocso miatt mutétre kerülo betegek összehasonlító vizsgálata.

INTRODUCTION: Barrett's esophagus (BE) is the only known precursor of adenocarcinoma occuring in the lower third of the esophagus. According to statistics, severity and elapsed time of gastroesophageal reflux disease (GERD) are major pathogenetic factors in the development of Barrett's esophagus. PATIENTS AND METHODS: In a retrospective study between 2001 and 2008, we compared the preoperative results (signs and sympthoms, 24 hour pH manometry, esophageal manometry, Bilitec) and treatment efficacy of 176 GERD patients and 78 BE patients, who have undergone laparoscopic Nissen procedure for reflux disease. RESULTS: The two groups of patients had similar demographic features, and elapsed time of reflux sympthoms were also equal. Both groups were admitted for surgery after a median time of 1.5 years (19.87 vs. 19.20 months) of ineffective medical (proton pump inhibitors) treatment. Preoperative functional tests showed a more severe presence of acid reflux in the BE group (DeMeester score 18.9 versus 41.9, p < 0.001). On the other hand, mano-metry - despite confirming lower esophageal sphincter (LES) damage - did not show difference between the two groups (12.10 vs. 12.57 mmHg, p = 0.892). We did not experience any mortality cases with laparoscopic antireflux procedures, although in two cases we had to convert during the operation (1 due to extensive adhesions, and 1 due to injury to the spleen). 3 months after the procedure - according to Visick score - both groups experienced a significant decrease, or lapse in reflux complaints (group I: 73%, group II: 81% of patients), LES functions improved (17.58 vs.18.70 mmHg), and the frequency and exposition of acid reflux decreased (DeMeester score 7.73 vs. 12.72). CONCLUSION: The severity of abnormal acid reflux occuring parallel with the incompetent function of the damaged LES triggers not only inflammation in the gastroesophageal junction (GEJ), but also metaplastic process, and the development of Barrett's esophagus. Laparoscopic Nissen procedure for reflux disease can further improve outcome among patients with GERD not responding to conservative therapy.

Impact of the Alvarado score on the diagnosis of acute appendicitis: comparing clinical judgment, Alvarado score, and a new modified score in suspected appendicitis: a prospective, randomized clinical trial.

BACKGROUND: The Alvarado score is a clinical scoring system used in the diagnosis of acute appendicitis. This study aimed to compare the reliability of the Alvarado score and clinical judgment and to refine the score to make it easier to use. METHODS: In this prospective, randomized study, patients presenting at the authors' outpatient department with suspected appendicitis during a 1-year period were assigned in weekly alternation to either group A or group B. The group A patients were treated on the basis of their Alvarado score, and the group B patients underwent treatment based on clinical judgment. The correctness of the methods was assessed by evaluation of the final histology. Statistical comparison of the data was performed using SPSS 20. RESULTS: The study investigated 269 patients (131 in group A and 138 in group B). The groups were homogeneous in terms of mean age, gender, body mass index, and American Society of Anesthesiologists score. The number of negative appendectomies was 12 (9.16%) in group A versus 5 (3.6%) in group B (p = 0.063). The clinical judgment had better specificity and sensitivity than the Alvarado score. For that reason, the specificity of the Alvarado score was refined using statistical methods, with weighting of certain clinical data and inclusion of new ones (e.g., ultrasound investigation). Consequently, the area under the curve by receiver operating characteristic analysis gradually increased, and the Alvarado score became more accurate. CONCLUSION: The study findings showed clinical judgment to be more reliable in the diagnosis of acute appendicitis than the Alvarado score, but the score is a useful diagnostic aid, especially for young colleagues. The use of the new scoring system has become easier. It includes fewer criteria as well as an important and sensitive predictor: the ultrasound investigation.