Surgical treatment of congenital pseudarthrosis of the clavicle: a report on 17 cases

Congenital pseudarthrosis of the clavicle (CPC) is a rare malformation of uncertain aetiopathogenesis, usually unilateral. Physical examination reveals swelling over the midportion of the clavicle, often asymptomatic; the diagnosis is confirmed by radiology. Treatment is controversial: for many authors the surgical indications are the presence of symptoms, functional impairment or cosmetic deformities. We present a retrospective analysis of 17 children with CPC treated in our institutions: 9 were treated with plate (P) and 8 with Kirschner wire (KW) fixation; a bone graft was used in 12 cases only. Five patients (4 P and 1 KW) needed a second surgical procedure. The surgical treatment led to a very good result in 7 cases, good in 4 cases, fair in 3 cases and poor in 3 other cases. We recommend early treatment of all patients with CPC with resection of the pseudarthrosis, autologous iliac bone grafting and internal fixation with Kirschner wires.     

Brain glucagon-like peptide-1 regulates arterial blood flow, heart rate, and insulin sensitivity

OBJECTIVE— To ascertain the importance and mechanisms underlying the role of brain glucagon-like peptide (GLP)-1 in the control of metabolic and cardiovascular function. GLP-1 is a gut hormone secreted in response to oral glucose absorption that regulates glucose metabolism and cardiovascular function. GLP-1 is also produced in the brain, where its contribution to central regulation of metabolic and cardiovascular homeostasis remains incompletely understood.RESEARCH DESIGN AND METHODS— Awake free-moving mice were infused with the GLP-1 receptor agonist exendin-4 (Ex4) into the lateral ventricle of the brain in the basal state or during hyperinsulinemic eu-/hyperglycemic clamps. Arterial femoral blood flow, whole-body insulin-stimulated glucose utilization, and heart rates were continuously recorded.RESULTS— A continuous 3-h brain infusion of Ex4 decreased femoral arterial blood flow and whole-body glucose utilization in the awake free-moving mouse clamped in a hyperinsulinemic-hyperglycemic condition, only demonstrating that this effect was strictly glucose dependent. However, the heart rate remained unchanged. The metabolic and vascular effects of Ex4 were markedly attenuated by central infusion of the GLP-1 receptor (GLP-1R) antagonist exendin-9 (Ex9) and totally abolished in GLP-1 receptor knockout mice. A correlation was observed between the metabolic rate and the vascular flow in control and Ex4-infused mice, which disappeared in Ex9 and GLP-1R knockout mice. Moreover, hypothalamic nitric oxide synthase activity and the concentration of reactive oxygen species (ROS) were also reduced in a GLP-1R–dependent manner, whereas the glutathione antioxidant capacity was increased. Central GLP-1 activated vagus nerve activity, and complementation with ROS donor dose-dependently reversed the effect of brain GLP-1 signaling on peripheral blood flow.CONCLUSIONS— Our data demonstrate that central GLP-1 signaling is an essential component of circuits integrating cardiovascular and metabolic responses to hyperglycemia.There is now compelling evidence supporting the interplay between metabolic and vascular diseases (1,2) in which neuronal circuits in the central nervous system seem to play a critical role in orchestrating the control of glucose homeostasis (3). We recently demonstrated that the central infusion of insulin decreased blood pressure and increased arterial blood flow and heart rate through a molecular mechanism depending on the synthesis of nitric oxide in the hypothalamus (4). Importantly, the central regulation of nitric oxide (NO) metabolism affected whole-body glucose utilization (5). This mechanism was impaired during high-fat diet–induced insulin resistance and diabetes and reverted upon central NO supplementation (4). These findings raise the possibility that signals from peripheral tissues, which act on the brain to control glucose metabolism, could also regulate vascular function.Enteroendocrine cells have important roles in regulating energy intake and glucose homeostasis through their actions on peripheral target organs, including the endocrine pancreas. Enteroendocrine cells secrete multiple hormones, including glucagon-like peptide (GLP)-1, which controls pancreatic endocrine secretion (6). GLP-1 is also a neuropeptide synthesized by neurons in the caudal regions of the nucleus of the solitary tract (NTS) (7,8). GLP-1 is released into the hypothalamus and controls food intake, blood pressure, and heart rate (9,10). Whereas most of the glucose-lowering actions of GLP-1 have been attributed to the direct effect of the hormone on the endocrine pancreas, i.e., to stimulation of insulin and inhibition of glucagon secretion, we demonstrated the importance of extra-pancreatic GLP-1 receptor–dependent control of insulin secretion (11) and whole-body glucose distribution (12). The infusion into the brain of the GLP-1 receptor antagonist exendin-9 (Ex9) inhibited insulin secretion induced by gut glucose (11). Conversely, central administration of the GLP-1 receptor agonist exendin-4 (Ex4) augmented intravenous glucose-stimulated insulin secretion to a level similar to that obtained during an intragastric glucose infusion (11). Our data suggested that the absorptive state was associated with the stimulation of the gut-to-brain axis leading to the activation of brain GLP-1 signaling and, consequently, to hyperinsulinemia. During the absorptive state, blood flow redistribution toward mesenteric organs is also observed, which has been proposed to favor nutrient redistribution into the liver (13). Importantly, stimulation of the central GLP-1 receptor increases blood pressure and heart rate and activates autonomic regulatory neurons (8,14,15). However, recently it has been shown that GLP-1 reduced islet blood flow after glucose administration (16). Therefore, the role of brain GLP-1 signaling also in the control of cardiovascular homeostasis remains incompletely understood.We have now pursued the importance of GLP-1 action in the central nervous system for control of cardiovascular function using studies in conscious free-moving mice. After central GLP-1 infusion, we simultaneously recorded femoral arterial blood flow, heart rate, and insulin and glucose sensitivity during hyperinsulinemic-euglycemic or hyperglycemic clamps. We now demonstrate that hypothalamic reactive oxygen and nitrogen species are controlled by brain GLP-1 and are essential for the coordinated regulation of metabolic and cardiovascular function.     

Where FoxP3-dependent regulatory T cells impinge on the development of inflammatory arthritis

OBJECTIVE: Regulatory T cells play a suppressive role in many autoimmune diseases and can potentially affect various steps in the progression of disease. The purpose of this study was to analyze the role of Treg cells in the control of arthritis development. METHODS: Using crosses and cell transfers, we tested the effect of the scurfy loss-of-function mutation of the Foxp3 gene in the K/BxN mouse model. In this model, arthritis develops as the result of the production of high levels of pathogenic autoantibodies. RESULTS: The absence of Treg cells in K/BxN mice led to faster and more aggressive arthritis. Strikingly, disease also spread to joints not normally affected in this model. The absence of Treg cells resulted in an acceleration of the immunologic phase of disease, with significantly earlier autoantibody production. However, the broadened spectrum of affected joints in Foxp3-mutant mice was not due to the earlier appearance of autoantibodies and could not be reproduced by increasing the anti-glucose-6-phosphate isomerase antibody load, which demonstrates an impact of Treg cells on effector phase manifestations. In addition, FoxP3+,CD25+ Treg cells accumulated in inflamed joints, even in nontransgenic animals. This preferential localization mimics that in human arthritides and indicates a preferential homing/retention of Treg cells to sites of inflammation. CONCLUSION: These results indicate that Treg cells play a role in antibody-mediated arthritis at several levels. Treg cells are involved in constraining the immune phase of disease, as well as limiting the articular damage provoked by the pathogenic autoantibodies in terms of severity and of the range of affected joints, which may contribute to the limited distal predominance of many arthritides.     

Donor-derived cells and human graft-versus-host disease of the skin

Graft-versus-host disease (GvHD)-induced apoptosis of the skin targets both epidermal keratinocytes and dermal endothelial cells. We studied the donor-versus-recipient origin of GvHD of these target cells in skin of 18 sex-mismatched hematopoietic stem-cell transplant (HSCT) recipients. Combining XY fluorescence in situ hybridization (FISH) and double immunostaining, and further 3D tissue Z-stack analysis, we found keratinocytes and endothelial cells of donor origin, but only in patients with GvHD. Using terminal dUTP nick-end labeling (TUNEL) assay on sister sections, we found a correlation between the numbers of chimeric and apoptotic epidermal and endothelial cells. Moreover, donor-derived cells were more numerous and preferentially distributed in the areas of severe GvHD damage in biopsies performed early in the course of GvHD, whereas they were less numerous and found in the whole epidermis in late biopsies. Because donor-derived cells were found at the site and at the time of maximum tissue damage, they could contribute to epidermal and microvessel repair.     

Deletions within the HSV-tk transgene in long-lasting circulating gene-modified T cells infused with a hematopoietic graft

In our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk(+))/neomycin phosphotransferase (NeoR(+))-expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell-depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk-polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel "transgene walking" PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti-HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell-depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.     

RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries

OBJECTIVE: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. METHODS: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118+/-15 microm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. RESULTS: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3+/-2.8 microm and 9.1+/-3.7 microm in +/+ and -/- mice, respectively, n = 5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26+/-13% merging at 25 mmHg versus 97+/-21% at 150 mmHg, n = 5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. CONCLUSION: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.     

Therapeutic strategies in oesophageal carcinoma: role of surgery and other modalities

Traditionally, surgery is considered the best treatment for oesophageal cancer in terms of locoregional control and long-term survival. However, survival 5 years after surgery alone is about 25%, and, therefore, a multidisciplinary approach that includes surgery, radiotherapy, and chemotherapy, alone or in combination, could prove necessary. The role of each of these treatments in the management of oesophageal cancer is under intensive research to define optimum therapeutic strategies. In this report we provide an update on treatment strategies for resectable oesophageal cancers on the basis of recent published work. Results of the latest randomised trials allow us to propose the following guidelines: surgery is the standard treatment, to be used alone for stages I and IIa, or possibly with neoadjuvant chemotherapy or chemoradiotherapy for stage IIb disease. For locally advanced cancers (stage III), neoadjuvant chemotherapy or chemoradiotherapy followed by surgery is appropriate for adenocarcinomas. Chemoradiotherapy alone should only be considered in patients with squamous-cell carcinomas who show a morphological response to chemoradiotherapy, and produces a similar overall survival to chemoradiotherapy followed by surgery, but with less post-treatment morbidity. Although the addition of surgery to chemotherapy or chemoradiotherapy could result in improved local control and survival, surgery should be done in experienced hospitals where operative mortality and morbidity are low. Moreover, surgery should be kept in mind as salvage treatment in patients with no morphological response or persistent tumour after definitive chemoradiotherapy.     

Nectin-4 is a new histological and serological tumor associated marker for breast cancer

Introduction

Breast cancer is a complex and heterogeneous disease at the molecular level. Evolution is difficult to predict according to classical histoclinical prognostic factors. Different studies highlight the importance of large-scale molecular expression analyses to improve taxonomy of breast cancer and prognostic classification. Identification of new molecular markers that refine this taxonomy and improve patient management is a priority in the field of breast cancer research. Nectins are cell adhesion molecules involved in the regulation of epithelial physiology. We present here Nectin-4/PVRL4 as a new histological and serological tumor associated marker for breast carcinoma.

Methods

Expression of Nectin-4 protein was measured on a panel of 78 primary cells and cell lines from different origins and 57 breast tumors by FACS analysis and immunohistochemistry (IHC), respectively. mRNA expression was measured by quantitative PCR. Serum Nectin-4 was detected by ELISA and compared with CEA and CA15.3 markers, on panels of 45 sera from healthy donors, 53 sera from patients with non-metastatic breast carcinoma (MBC) at diagnosis, and 182 sera from patients with MBC. Distribution of histological/serological molecular markers and histoclinical parameters were compared using the standard Chi-2 test.

Results

Nectin-4 was not detected in normal breast epithelium. By contrast, Nectin-4 was expressed in 61% of ductal breast carcinoma vs 6% in lobular type. Expression of Nectin-4 strongly correlated with the basal-like markers EGFR, P53, and P-cadherin, and negatively correlated with the luminal-like markers ER, PR and GATA3. All but one ER/PR-negative tumors expressed Nectin-4. The detection of Nectin-4 in serum improves the follow-up of patients with MBC: the association CEA/CA15.3/Nectin-4 allowed to monitor 74% of these patients compared to 67% with the association CEA/CA15.3. Serum Nectin-4 is a marker of disease progression, and levels correlate with the number of metastases (P = 0.038). Serum Nectin-4 is also a marker of therapeutic efficiency and correlates, in 90% of cases, with clinical evolution.

Conclusion

Nectin-4 is a new tumor-associated antigen for breast carcinoma. Nectin-4 is a new bio-marker whose use could help refine breast cancer taxonomy and improve patients' follow-up. Nectin-4 emerges as a potential target for breast cancer immunotherapy.