Tuberin,the tuberous sclerosis complex 2 tumor suppressor gene product,regulates Rho activation,cell adhesion and migration

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors of the brain, kidney, heart, retina, and skin. TSC is caused by mutations in either TSC1 or TSC2, both of which are tumor suppressor genes. Hamartin, the protein product of TSC1, was found to interact with the ezrin-radixin-moesin family of cytoskeletal proteins and to activate the small GTPase Rho. To determine whether tuberin, the TSC2 product, can also activate Rho, we stably expressed full-length human tuberin in two cell types: MDCK cells and ELT3 cells. ELT3 cells lack endogenous tuberin expression. We found that expression of human tuberin in both MDCK and ELT3 cells was associated with an increase in the amount of Rho-GTP, but not in Rac1-GTP or cdc42-GTP. Tuberin expression increased cell adhesion in both cell types, and decreased chemotactic cell migration in ELT3 cells. In MDCK cells, there was a decrease in the amount of total Focal Adhesion Kinase (FAK) and an increase in the fraction of phosphorylated FAK. These findings demonstrate for the first time that tuberin activates Rho and regulates cell adhesion and migration. Pathways involving Rho activation may have relevance to the clinical manifestations of TSC, including pulmonary lymphangioleiomyomatosis.     

Hypoxia and an angiogenic response in the partially obstructed rat bladder

Previous molecular and blood flow studies performed on animal models of partial bladder outlet obstruction (PBOO) caused us to propose that bladder hypoxia/ischemia was a significant effector of the cellular and functional changes that occur in the bladder as a result of this condition. To confirm the occurrence of hypoxia in the partially obstructed bladder, we obtained rat bladders at increasing intervals following PBOO and measured biomarkers of hypoxia (intracellular formation of hypoxyprobe-1 adducts and expression of hypoxia inducible factor-1 alpha [HIF-1 alpha] protein) and whether such hypoxia might elicit an angiogenic response in the tissue. Rats receiving PBOO or controls were treated with hypoxyprobe-1 at increasing intervals subsequent to surgery and their bladders were sectioned and immunostained using an antibody that detects hypoxyprobe-1 adducts. Control rat bladders were unstained, whereas intense, but regionally restricted, hypoxyprobe-1 immunostaining was detected in all obstructed bladders in a unique pattern that changed over time. Proteins were extracted from bladders removed from similarly treated rats and were analyzed for the expression of the HIF-1 alpha protein as well as for expression of angiogenic regulatory factors (vascular endothelial growth factor, angiopoietin-1, and endostatin) using Western blotting techniques. HIF-1 alpha protein was not expressed in control bladders, however, the protein was highly up-regulated over the 2-week period after PBOO. Likewise, the expression of vascular endothelial growth factor (a downstream target of HIF-1 alpha action) and angiopoietin-1 was also up-regulated in obstructed bladders confirming an angiogenic response to this hypoxia. Enigmatically, however, expression of the antiangiogenic molecule endostatin was also up-regulated by chronic PBOO. These results further support the concept that hypoxia is involved in the cellular remodeling as well as in the progressive functional impairment exhibited by the urinary bladder after PBOO.     

Combination docetaxel (Taxotere), fluorouracil, and leucovorin (TFL), as first-line chemotherapy in advanced gastric cancer: a Hellenic Cooperative Oncology Group phase II study

Background We assessed the efficacy and safety profile of a docetaxel (Taxotere; Sanofi-Aventis, France), fluorouracil (FU), and leucovorin (LV) combination (TFL), as first-line chemotherapy in patients with advanced gastric cancer. Methods Fifty-eight patients with advanced gastric cancer were entered in this phase II study. The chemotherapy regimen (TFL) was administered in an outpatient setting as follows: docetaxel 7mg/m² on day 1 and FU 50mg/m² and LV 30mg/m² on days 1 to 3, every 3 weeks for six cycles. Results On an intent-to-treat basis, 4 complete (7%) and 11 partial responses (19%) were observed, with an objective overall response rate of 26%; in addition, 22 patients (38%) had stable and 15 (26%) had progressive disease. For 6 (10%) patients, response could not be evaluated. Responses were noted at all metastatic sites. With a median follow-up of 55 months, median survival was 9 months; median time to progression, 5.9 months; and median duration of response, 10 months. Toxicity was manageable and no toxic death was reported. Neutropenia was the most frequent severe toxicity and occurred in 30% of the patients. The main non-hematologic toxicities were alopecia (76%), diarrhea (30%), and stomatitis (30%). Conclusion The results of this phase II study seem to indicate that the TFL regimen has moderate activity in patients with advanced gastric cancer, with acceptable toxicity.     

Hydrocephalus and aquaporins: lessons learned from the bench

Purpose  

Hydrocephalus is a common disorder of defective cerebrospinal fluid (CSF) turnover. The identification of the aquaporin water channels (AQPs) led to the study of their role in the composition of biological fluids including CSF. The purpose of this study is to review the potential role of aquaporins in the pathogenesis, compensation, and possibly treatment of hydrocephalus.     

Lymph node metastasis of carcinomas of transverse colon including flexures. Consideration of the extramesocolic lymph node stations

Purpose

Complete mesocolic excision (CME) is nowadays state of the art in the treatment of colon cancer. In cases of carcinoma of transverse colon and of both flexures an extramesocolic lymph node metastasis can be found in the infrapancreatic lymph node region (ILR) and across the gastroepiploic arcade (GLR). These direct metastatic routes were not previously systematically considered. In order to validate our hypothesis of these direct metastatic pathways and to obtain evidence of our approach of including dissection of these areas as part of CME, we initiated a prospective study evaluating these lymph node regions during surgery.

Methods

Forty-five consecutive patients with primary tumour manifestation in transverse colon and both flexures between May 2010 and January 2013 were prospectively analyzed. Patients were followed up for at least 6 months. Mode of surgery, histopathology, morbidity and mortality were evaluated.

Results

Twenty-six patients had a carcinoma of transverse colon, 16 patients one of hepatic flexure and four patients one of splenic flexure. The median lymph node yield was 40. Occurrence of lymph node metastasis in ILR was registered in five patients and in GLR in four patients. The mean lymph node ratio was 0.085. Postoperative complications occurred in nine patients, and postoperative mortality was 2 %.

Conclusions

We were able to demonstrate this novel metastatic route of carcinomas of the transverse colon and of both flexures in ILR and GLR. These could be considered as regional lymph node regions and have to be included into surgery for cancer of the transverse colon including both flexures.     

Differential immunogenicity and allergenicity of native and recombinant human lactoferrins: Role of glycosylation

Human native milk lactoferrin (LF) and recombinant forms of lactoferrin (rLF) are available with identical aa sequences, but different glycosylation patterns. Native lactoferrin (NLF) possesses the intrinsic ability to stimulate vigorous IgG and IgE antibody responses in BALB/c mice, whereas recombinant forms (Aspergillus or rice) are 40‐fold less immunogenic and 200‐fold less allergenic. Such differences are independent of endotoxin or iron content and the glycans do not contribute to epitope formation. A complex glycoprofile is observed for NLF, including sialic acid, fucose, mannose, and Lewis (Le)^x structures, whereas both rLF species display a simpler glycoprofile rich in mannose. Although Le^x type sugars play a Th2‐type adjuvant role, endogenous expression of Le^x on NLF did not completely account for the more vigorous IgE responses it provoked. Furthermore, coadminstration of rLF downregulated IgE and upregulated IgG2a antibody responses provoked by NLF, but was without effect on responses to unrelated peanut and chicken egg allergens. These results suggest glycans on rLF impact the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation, and the balance between Th1 and Th2 responses.     

The use of surface immobilization of P-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling

Mesenchymal stem/stromal cells (MSCs) are an important candidate for cell-based therapy since they can be easily isolated and expanded, secrete beneficial paracrine factors, and differentiate into multiple lineages. Since the endothelium at sites of injury and inflammation often express adhesion molecules belonging to the selectin family, methods to endow MSCs with selectin-ligands can enhance the efficacy of cell delivery and tissue engraftment. Here, we describe a construct 19Fc[FUT7⁺], where the first 19 amino acids of the pan-selectin ligand PSGL-1 (P-selectin glycoprotein ligand-1) was fused to a human IgG1. When expressed in HEK293T cells over-expressing the α(1,3)fucosyltransferase FUT7, 19Fc[FUT7⁺] is decorated by a core-2 sialyl Lewis-X sialofucosylated O-glycan. The non-covalent coupling of this protein onto MSC surface using palmitated protein G (PPG) enhanced cell binding to E- and P-selectin under hydrodynamic shear, without altering MSC multipotency. MSCs functionalized with 19Fc[FUT7⁺] were captured/tethered onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm². Once captured, the cells rolled robustly up to the highest shear stress tested, 10 dyn/cm². Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a ‘glycan engineering’ strategy to enable leukocyte-like capture and rolling.