Stomach engineering: region-specific characterization of the decellularized porcine stomach.

PURPOSE: Patients affected by microgastria, severe gastroesophageal reflux, or those who have undergone subtotal gastrectomy, have commonly described reporting dumping syndromes or other symptoms that seriously impair the quality of their life. Gastric tissue engineering may offer an alternative approach to treating these pathologies. Decellularization protocols have great potential to generate novel biomaterials for large gastric defect repair. There is an urgency to define more reliable protocols to foster clinical applications of tissue-engineered decellularized gastric grafts. METHODS: In this work, we investigated the biochemical and mechanical properties of decellularized porcine stomach tissue compared to its native counterpart. Histological and immunofluorescence analyses were performed to screen the quality of decellularized samples. Quantitative analysis was also performed to assess extracellular matrix composition. At last, we investigated the mechanical properties and cytocompatibility of the decellularized tissue compared to the native. RESULTS: The optimized decellularization protocol produced efficient cell removal, highlighted in the absence of native cellular nuclei. Decellularized scaffolds preserved collagen and elastin contents, with partial loss of sulfated glycosaminoglycans. Decellularized gastric tissue revealed increased elastic modulus and strain at break during mechanical tensile tests, while ultimate tensile strength was significantly reduced. HepG2 cells were seeded on the ECM, revealing matrix cytocompatibility and the ability to support cell proliferation. CONCLUSION: Our work reports the successful generation of acellular porcine gastric tissue able to support cell viability and proliferation of human cells.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Intussusception and COVID-19 in Infants: Evidence for an Etiopathologic Correlation.

Nonrespiratory conditions related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been largely described. Ileocolic intussusception has been reported in association with SARS-CoV-2 infection in 10 children, raising the possibility of an etiopathologic role for the virus, but none of these cases documented tissue pathology that would have supported SARS-CoV-2 intestinal inflammation. We report 2 cases of intussusception in patients with SARS-CoV-2 infection who were treated at different pediatric tertiary centers in Europe and provide evidence of the presence of the virus in mesenteric and intestinal tissues of the patients.

Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase, and vitamin D receptor mRNA in normal and malignant breast tissue.

Vitamin D has anti-proliferative and proapoptotic effects on several cell types, including breast cancer cells. There have been no studies quantifying the expression of the enzyme 25-hydroxyvitamin D-1-alpha-hydroxylase (1alphaOHase), which converts 25-hydroxyvitamin D to its active metabolite, in breast tissue. We performed real-time RT-PCR to quantity 1alphaOHase and vitamin D receptor (VDR) mRNA in samples of breast cancer, adjacent non-cancerous tissue and normal breast tissue. 1alphaOHase and VDR mRNA were universally expressed, however, 1alphaOHase expression was significantly down-regulated in adjacent non-cancerous tissue from women with breast cancer in comparison to individuals without cancer. VDR was also up-regulated in breast tumours. The vitamin D axis expression in the breast suggests a role for its actions in normal tissue homeostasis and breast cancer pathogenesis. The decreased expression of 1alphaOHase in normal tissue from women with breast cancer may be important in their predisposition to the development of cancer.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours.

OBJECTIVES: Somatostatin (SST) analogues play an important role in the medical management of somatotroph pituitary adenomas and new agonists have the potential to be effective in a wider group of pituitary and other tumours. The anti-proliferative effect of SST occurs through multiple mechanisms, one of which is cell-cycle arrest, where p27, a cyclin-dependent kinase inhibitor, is an important regulator. We hypothesised that SST may upregulate p27 protein levels and downregulate the MAP kinase pathway in these tumours. METHODS: Human pituitary adenoma cells and rat pituitary cell line (GH3) were cultured and treated in vitro with octreotide and the broad-spectrum SST agonist SOM230 (pasireotide). Immunoblotting for p27 and phospho-ERK (pERK) was performed and proliferation assessed by [3H]-thymidine incorporation. Histological samples from acromegalic patients treated with octreotide before surgery were immunostained for p27 and compared to samples from untreated patients matched for sex, age, tumour size, extension and invasiveness. RESULTS: We detected upregulation of p27 protein levels with SST analogue treatment in vitro in human pituitary adenoma samples. pERK1/2 was inhibited by SST analogues in both the human samples and GH3 cells. SST and its analogues inhibited the proliferation of GH3 cells. p27 immunostaining was stronger in samples from patients with longer preoperative octreotide treatment (more than 6 months) than in samples from patients with shorter treatment periods. CONCLUSIONS: This study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours, where the MAP kinase pathway is overactivated.

Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway.

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.

The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes.

BACKGROUND: Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and therefore they are functional candidates for genes carrying susceptibility alleles for type 2 diabetes. METHODS: We assessed common genetic variation of the ghrelin (GHRL; five single nucleotide polymorphisms (SNP)) and the ghrelin-receptor (GHSR) genes (four SNPs) in 610 Caucasian patients with type 2 diabetes and 820 controls. In addition, promoter reporter assays were conducted to model the regulatory regions of both genes. RESULTS: Neither GHRL nor GHSR gene SNPs were associated with type 2 diabetes. One of the ghrelin haplotypes showed a marginal protective role in type 2 diabetes. We observed profound differences in the regulation of the GHRL gene according to promoter sequence variants. There are three different GHRL promoter haplotypes represented in the studied cohort causing up to 45% difference in the level of gene expression, while the promoter region of GHSR gene is primarily represented by a single haplotype. CONCLUSION: The GHRL and GHSR gene variants are not associated with type 2 diabetes, although GHRL promoter variants have significantly different activities.