Semiparametric M-quantile regression for count data.

Lung cancer incidence over 2005-2010 for 326 Local Authority Districts in England is investigated by ecological regression. Motivated from mis-specification of a Negative Binomial additive model, a semiparametric Negative Binomial M-quantile regression model is introduced. The additive part relates to those univariate or bivariate smoothing components, which are included in the model to capture nonlinearities in the predictor or to account for spatial dependence. All such components are estimated using penalized splines. The results show the capability of the semiparametric Negative Binomial M-quantile regression model to handle data with a strong spatial structure.

A new clinical approach: use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses.

AIMS: In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS: Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS: The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE: This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

Outcomes of unrelated cord blood transplants and allogeneic-related hematopoietic stem cell transplants in children with high-risk acute lymphocytic leukemia.

Acute lymphocytic leukemia (ALL) is a common indication for hematopoietic stem cell transplantation (HSCT) in children. Use of unrelated cord blood (UCB) has become increasingly popular as a stem cell source, given the rapid availability and decreased GVHD potential. Publications describing outcomes of children with leukemia who underwent UCB transplants have compared them to those having received unrelated donor marrow transplants. Results are similar. We compared our outcomes using UCB vs allogeneic-related hematopoietic stem cells in pediatric ALL patients since 1992. A total of 49 patients were analyzed. All patients were either in CR1 with high-risk features (n=21) or in CR2 (n=28) with initial remission less than 36 months. Patients received myeloablation with fractionated total body irradiation, cyclophosphamide, and etoposide and GVHD prophylaxis with cyclosporine and methotrexate. Antithymocyte globulin was added for UCB recipients to address the HLA differences. In all, 23 patients underwent allogeneic -related HSCT and 26 underwent UCB transplantation. Other than increased time to engraftment for UCB recipients, results are equivalent. The 3-year overall survival is 64% and 3-year event-free survival is 60% for both groups. Rates of GVHD and transplant-related mortality are also equivalent. UCB is a reasonable option for children with ALL who are referred for HSCT.

Role of GST P1-1 in mediating the effect of etoposide on human neuroblastoma cell line Sh-Sy5y.

The oxidative stress could have a dual action on glutathione S-transferase (GST) P1-1 metabolism: transcriptional induction and/or polymerization. The former should represent a form of adaptation to oxidative stress and contribute to protect the cell, the latter one should activate apoptosis via c-Jun N-terminal kinase (JNK). We studied the effect of etoposide on human neuroblastoma cell line SH-SY5Y and on an etoposide-resistant clone to investigate whether a pleiotropic effect of etoposide on the redox status of the cell exists which is able to interfere with apoptosis through the GST P1-1 system. Etoposide treatment was able to induce GST P1-1 polymerization and activation of apoptosis. The data obtained from our etoposide-resistant clone and the possibility to reverse the sensitive phenotype to a resistant one by means of hexyl-glutathione preincubation, seem to suggest that cellular levels of glutathione have a key role in protecting GST P1-1 by oxidation and consequently the cell's decision between life and death.

Transglutaminase 5 cross-links loricrin, involucrin, and small proline-rich proteins in vitro.

Transglutaminases (TGases) are seven enzymes, cross-linking proteins by gamma-glutamil-epsilon-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and Delta3 (deletion of exon 3), we identified two new splicing variants, Delta11 and Delta3Delta11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, Delta3, Delta11, and Delta3Delta11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and Delta11 are active, whereas Delta3 and Delta3Delta11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitro cross-linking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentin in vitro.

High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.

Pigment epithelium-derived factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell antitumor activity.

Neuroblastoma is notable for its cellular heterogeneity and unpredictable outcome. Tumors are a variable mixture of primitive malignant neuroblasts, more differentiated ganglionic cells, Schwann and endothelial cells. Although often fatal, neuroblastomas can spontaneously regress, possibly due to favorable autocrine and paracrine interactions among these cells. Here, pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis and inducer of neural differentiation, is shown to be produced by ganglionic cells and Schwann cells, but not by more primitive tumor cells. Although undifferentiated neuroblastoma tumor cell secretions were angiogenic primarily due to vascular endothelial growth factor, secretions of Schwann cells were anti-angiogenic due to PEDF. In addition, PEDF was the major factor responsible for Schwann cell's ability to induce tumor cell differentiation in vitro and recombinant PEDF had the same effect in vitro and in vivo. Both the growth and the survival of Schwann cells were enhanced by PEDF. Thus PEDF may serve as a multifunctional antitumor agent in neuroblastomas, inhibiting angiogenesis while promoting the numbers of Schwann cells and differentiated tumor cells that in turn produce PEDF, suggesting that its clinical administration could stimulate a multifaceted antitumor feedback loop with the potential to limit and possibly regress tumor growth.

The common Arg972 polymorphism in insulin receptor substrate-1 causes apoptosis of human pancreatic islets.

Molecular scanning of human IRS-1 gene revealed a common polymorphism causing Gly-->Arg972 change. Diabetic and pre-diabetic carriers of Arg972 IRS-1 are characterized by low fasting levels of insulin and C-peptide. To investigate directly whether the Arg 972 IRS-1 affects human islet cells survival, we took advantage of the unique opportunity to analyze pancreatic islets isolated from three donors heterozygous for the Arg972 and six donors carrying wild-type IRS-1. Islets from carriers of Arg972 IRS-1 showed a two-fold increase in the number of apoptotic cells as compared with wild-type. IRS-1-associated PI3-kinase activity was decreased in islets from carriers of Arg972 IRS-1. Same results were reproduced in RIN rat b-cell lines stably expressing wild-type IRS-1 or Arg972 IRS-1. Using these cells, we characterized the downstream pathway by which Arg972 IRS-1 impairs b-cell survival. RIN-Arg972 cells exhibited a marked impairment in the sequential activation of PI3-kinase, Akt, and BAD as compared with RI N-WT. Impaired BAD phosphorylation resulted in increased binding to Bcl-XL instead of 14-3-3 protein, thus sequestering the Bcl-XL antiapoptotic protein to promote survival. Both caspase-9 and caspase-3 activities were increased in RIN-Arg972 cells. The results show that the common Arg972 polymorphism in IRS-1 impairs human b-cell survival and causes resistance to antiapoptotic effects of insulin by affecting the PI3-kinase/Akt survival pathway. These findings establish an important role for the insulin signaling in human b-cell survival and suggest that genetic defects in early steps of insulin signaling may contribute to b-cell failure.

Distinct properties of fenretinide and CD437 lead to synergistic responses with chemotherapeutic reagents.

The RARbeta/gamma-selective retinoids fenretinide and CD437 induce caspase-dependent apoptosis but generate free radicals independently of caspases. Apoptosis, but not free radical generation, induced by these retinoids is inhibited by RARbeta/gamma-specific antagonists. Both fenretinide and CD437 induce apoptosis synergistically with cisplatin, carboplatin, or etoposide. However, antioxidants inhibit this synergy to the level obtained with chemotherapeutic drugs alone, and this implies that free radical generation is important in the synergistic response. Since apoptosis induced by fenretinide or CD437 is mediated by apoptotic pathways involving RARs and/or mitochondria and differs from mechanisms of chemotherapy-induced apoptosis this may explain the strong synergistic response seen between these synthetic retinoids and chemotherapeutic drugs. These results suggest that fenretinide or CD437 may be useful adjuncts to neuroblastoma therapy.

Synergistic induction of apoptosis of neuroblastoma by fenretinide or CD437 in combination with chemotherapeutic drugs.

Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.

Effector mechanisms of fenretinide-induced apoptosis in neuroblastoma.

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Activation of different lipoxygenase isozymes induces apoptosis in human erythroleukemia and neuroblastoma cells.

We investigated the ability of different hydroperoxides generated by lipoxygenase isozymes to induce programmed cell death (PCD) in human cells. Erythroleukemia K562 and neuroblastoma CHP100 cells were used, because they showed high basal activity of lipoxygenase. The hydroperoxides generated by 5-, 12-, or 15-lipoxygenases from linoleate, linolenate, or arachidonate, and the corresponding hydroxides, were able to induce PCD in both cell types, in a concentration- and time-dependent manner. After 24 h, K562 and CHP100 cells showed 2.5- to 3.5-fold more apoptotic bodies than the untreated controls. PCD elicited by lipoxygenase products was independent of intracellular glutathione concentration, and did not require mRNA transcription or protein synthesis. On the other hand, lipoxygenase products evoked an immediate and sustained rise in cytoplasmic calcium (within seconds), followed by mitochondrial uncoupling (within hours). Unlike the hydro(pero)xides, the terminal products of the arachidonate cascade (i.e., leukotrienes, prostaglandins and thromboxane) were not cytotoxic.

[Locoregional anesthesia in inguinal hernia surgery]. / L'anestesia locoregionale nella chirurgia dell'ernia inguinale.

BACKGROUND: It is a current opinion that local anesthesia (LA) is the primary choice in surgical treatment of the inguinal region, particularly herniorrhaphy. The LA technique personally used for herniorrhaphy is described: it consists of iliohypogastric, ilioinguinal and genito-femoral nerve blocks, and incision line anesthetic infiltration. METHODS: From January 1998 to April 1999, 95 patients underwent inguinal herniorrhaphy employing LA: 77 (81%) in elective surgery, 18 (19%) in emergency; 2 cases with bilateral hernia (97 total LA procedures). RESULTS: Partial success was obtained in only 8 cases (8.4%), which required an association with a hypnotic drug ("blended anesthesia": propofol or midazolam): there were no cases of conversion to general anesthesia. Specific complications of local anesthetic drugs infiltration developed in 8 cases on 97 LA procedures (8.2%), but none required reoperation: 6 inguinal hematomas, 1 female external genitalia hematoma, 1 hematocele. CONCLUSIONS: In conclusion, it is stressed that LA is the technique of choice in herniorrhaphy and surgery of other inguinal pathologies, associating high success rates, rare complications and rapid dismissal: this allows for easy management of the patients and a very important reduction of sanitary costs. The association of LA-hypnotic drugs (blended anesthesia) represents another important resource, since it avoids general anesthesia in many cases and allows a rapid psychophysical recovery.

Induction of neuronal differentiation by p73 in a neuroblastoma cell line.

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.

Rapid growth of cutaneous metastases after surgical resection of thrombospondin-secreting small blue round cell tumor of childhood.

In animal models, the importance of tumor-derived antiangiogenic factors in controlling metastases has been demonstrated by the growth acceleration of distant metastases after surgical excision of a primary tumor mass. We report the case of an infant who developed rapidly growing cutaneous metastases after surgical resection of a neoplasm of an upper extremity. The tumor was undifferentiated, with some morphological features of primitive neuroectodermal tumor. To test the possibility that the primary tumor was secreting an angiogenic inhibitor, cells from the primary tumor were grown in culture, and the culture medium was tested with an in vitro endothelial cell migration assay and Western blot. The cultured cells secreted sufficiently high levels of an angiogenic inhibitor to overcome the inducing ability of vascular endothelial growth factor and basic fibroblast growth factor. One of the secreted proteins was thrombospondin-1, a potent antiangiogenic glycoprotein. The rapid dissemination of distant metastases after resection of the primary tumor in this case suggests that tumor-derived angiogenic inhibitors are important in maintaining the local net balance of angiogenic mediators controlling the growth of micrometastasis.

Activation of nitric oxide synthase is involved in tamoxifen-induced apoptosis of human erythroleukemia K562 cells.

Tamoxifen induces apoptosis (programmed cell death) in human erythroleukemia K562 cells. Nitric oxide synthase activity and expression increased in apoptotic cells by 315% and 280%, respectively, compared to controls. The specific inhibitor of nitric oxide synthase, L-NAME, protected K562 cells from tamoxifen-induced apoptosis, whereas the nitric oxide donor, sodium nitroprusside (SNP), potentiated the apoptotic effect of the drug. In addition, 5-lipoxygenase was activated by tamoxifen and the specific enzyme inhibitor, MK886, protected K562 cells against the drug. Conversely, the 5-lipoxygenase product, 5-hydroperoxyeicosatetraenoic acid, enhanced the tamoxifen-induced apoptosis. Finally, tamoxifen altered also membrane properties of K562 cells.