Post-transplant lymphoproliferative disorders in children: the role of chemotherapy in the era of rituximab.

PTLD are the most frequent neoplasms in children postorgan transplantation. We describe our experience in the treatment of 14 children (three with early and 11 with late-onset disease) treated with a step-wise protocol developed at our institution. Treatment consisted of reducing immunosuppressants, followed by rituximab and chemotherapy if required. Rituximab, incorporated into the protocol in 2001, has been determinant for the total chemotherapy burden patients need to achieve remission. In seven patients who did not receive rituximab, anthracycline total dose ranged from 160 to 240 mg/m(2), while only one of the patients receiving rituximab required DOXO (range: 0-120 mg/m(2)) (p = 0.003). The use of alkylating agents was also notably lower in patients receiving rituximab (median dose = 1200 mg/m(2)) compared with those who did not receive rituximab (median dose = 5800 mg/m(2)) (p = 0.006). Twelve patients are in remission and two died, one from refractory disease and the other from septic shock. Two-year OS and EFS were 85.7% and 57%, respectively. In conclusion, our experience with the use of rituximab in children with PTLD after solid organ transplantation appeared to be associated with a lesser requirement for alkylating agents and anthracyclines compared with historical subjects, suggesting a reduction in the side effects of these agents.

Long-term results of the Spanish Protocol SO-95 for the treatment of non-metastatic high-grade osteosarcoma of the extremities in children

INTRODUCTION: The long-term results of the Spanish Study Protocol SEOP-SO-95 for treatment of localised osteosarcoma of the extremities in children were evaluated. PATIENTS AND METHODS: One hundred consecutive patients under 18 years of age from 22 institutions were enrolled from January 1995 to December 2000. Immunohistochemical expression of p53, HER/erbB-2 and P-glycoprotein were retrospectively studied in 27 patients. Treatment consisted of: preoperative chemotherapy with doxorubicin, cisplatin, high-dose methotrexate with leucovorin rescue and ifosfamide for 14 weeks; surgery of primary tumour in week 16; postoperative chemotherapy with the above-mentioned drugs for 25 weeks. RESULTS: With a median follow-up of 124 months (range 84-158 months), 69 patients (69%) were continuously event-free survivors; the 10-year probability of event-free survival (EFS) was 62%. Conservative surgery was performed in 85% of patients. Twenty-six patients had local recurrence or distant relapse. The median time to recurrence/ relapse was 27 months (range 17-93 months). The local recurrence rate was 7% (7 of the 100 patients); 4 had wide surgical margins, 2 marginal and 1 intralesional. Four patients died as a result of chemotherapy-related toxicity and 1 developed a second neoplasia (acute myeloid leukaemia). p53 expression and HER2/erbB-2 expression showed no effect on survival or EFS. CONCLUSIONS: This therapeutic protocol achieved good oncologic and orthopaedic results. We observed a significant treatment-related toxicity (AU)

The tumor suppressor protein PML controls apoptosis induced by the HIV-1 envelope.

Promyelomonocytic leukemia (PML) is a prominent oncosuppressor whose inactivation is involved in the pathogenesis of hematological and epithelial cancers. Here, we report that PML aggregated in nuclear bodies in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. PML aggregation occurred after the fusion of nuclei (karyogamy) within syncytia but before the apoptotic program was activated. The aggregation of PML was detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Using a range of specific inhibitors of PML (the oncogenic PML/RARalpha fusion product or specific small interfering RNAs), we demonstrated that, in Env-elicited syncytia, PML was required for activating phosphorylation of ataxia telangiectasia mutated (ATM), which colocalized with PML in nuclear bodies, in a molecular complex that also involved topoisomerase IIbeta-binding protein 1. PML knockdown thus inhibited the ATM-dependent DNA damage response that culminates in the activation of p53, p53-dependent transcription of pro-apoptotic genes and cell death. Infection of CD4-expressing cells with HIV-1 also induced syncytial apoptosis, which could be suppressed by inhibiting PML. Altogether, these data indicate that PML activation is a critical early event that participates in the apoptotic demise of HIV-1-elicited syncytia.

Localization of alpha-endosulphine in pancreatic somatostatin delta cells and expression during rat pancreas development.

AIMS/HYPOTHESIS: alpha-Endosulphine, a protein that belongs to the cAMP-regulated-phosphoprotein family, has been reported to modulate insulin secretion in vitro through interaction with the pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channel. In this study, we analysed the tissue distribution of alpha-endosulphine and determined its pancreatic cellular localization. METHODS: Quantitative tissue distribution of alpha-endosulphine was studied by RIA on tissue extracts and cellular/subcellular localization was done using immunocytochemistry, morphometry and western blot analysis. alpha-Endosulphine and somatostatin release from RINT-3 somatostatin-secreting cells was quantified by RIA. RESULTS: alpha-Endosulphine, concentrated particularly in the central nervous system, was also detected in a wide variety of tissues including the pancreas. Immunohistochemistry analysis of adult rat pancreatic sections showed that alpha-endosulphine localized in somatostatin delta cells, where its expression increased during post-natal development. Immunoreactive cells were detected from foetal age E19, and the number of somatostatin cells co-expressing alpha-endosulphine increased with developmental age from E19 until adult. alpha-Endosulphine, highly expressed in the cytoplasm of RINT3 somatostatin-secreting cell line, was recovered in the particulate fraction of RINT3 cell extracts but was not co-secreted with somatostatin. CONCLUSION/INTERPRETATION: alpha-Endosulphine is expressed in all tissues tested including pancreas and is also detected in plasma. Pancreatic alpha-endosulphine is specifically localized in somatostatin delta cells. This cytosolic protein is not co-secreted with somatostatin and could be physically associated with particulate components of the cells. These findings are not in favour of an endocrine/paracrine effect of alpha-endosulphine on the beta-cell K(ATP) channel.

Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes.

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.

Insulin production by engineered muscle cells.

Type 1 diabetic patients depend dramatically on insulin replacement therapy, which involves the administration of intermediate- or long-acting insulin, together with short-acting insulin to mimic physiological insulin profiles. However, the delayed-action preparations available are not generally able to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stably transfected with a chimeric gene obtained by linking the myosin-light chain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm). When differentiated, C2C12Insm myotube cells expressed high levels of insulin mRNA and protein, whereas no insulin was detected in myoblast cells. HPLC fractionation of culture medium and cell extracts from differentiated C2C12Insm cells revealed that about 90% of the proinsulin was processed to mature insulin. In addition, these cells released significant levels (about 100 microU/10(6) cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the differentiated C2C12Insm cells and was able to block the expression of the endogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepatoma cells. Furthermore, when C2C12Insm myoblast cells were transplanted into diabetic mice an increase in insulinemia and a decrease in hyperglycemia were observed. Thus, our results suggest that the use of engineered myotube cells continuously secreting a defined level of insulin might be a useful approach to improve the efficacy of insulin injection treatment.

Characterization of low-affinity binding sites for glibenclamide on the Kir6.2 subunit of the beta-cell KATP channel.

The ATP-sensitive K+ channel, an octameric complex of two structurally unrelated types of subunits, SUR1 and Kir6.2, plays a central role in the physiological regulation of insulin secretion. The sulfonylurea glibenclamide, which trigger insulin secretion by blocking the ATP-sensitive K+ channel, interacts with both high and low affinity binding sites present on beta-cells. The high affinity binding site has been localized on SUR1 but the molecular nature of the low affinity site is still uncertain. In this study, we analyzed the pharmacology of glibenclamide in a transformed COS-7 cell line expressing the rat Kir6.2 cDNA and compared with that of the MIN6 beta cell line expressing natively both the Kir6.2 and the SUR1 subunits. Binding studies and Scatchard analysis revealed the presence of a single class of low affinity binding sites for glibenclamide on the COS/Kir6.2 cells with characteristics similar to that observed for the low affinity site of the MIN6 beta cells.

alpha-Endosulfine, a new entity in the control of insulin secretion.

ATP-dependent potassium (K ATP) channels occupy a key position in the control of insulin release from the pancreatic beta cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from beta cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called 'endosulfines.' In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, alpha-endosulfine, and discuss its possible role in the physiology of the beta cell as well as in pathology.

Cloning and tissue distribution of a new rat olfactory receptor-like (OL2).

Polymerase chain reaction (PCR) was used to clone an intronless cDNA encoding a new member (named OL2) of the G protein-coupled receptor superfamily. The coding region of the rat OL2 receptor gene predicts a seven transmembrane domain receptor of 315 amino acids. OL2 has 46.4 percent amino acid identity with OL1, an olfactory receptor expressed in the developing rat heart, and slightly lower percent indentities with several other olfactory receptors. PCR analysis reveals that the transcript is present mainly in the rat spleen and in a mouse insulin-secreting cell line (MIN6). No correlation was found between the tissue distribution of OL2 and that of the olfaction-related GTP-binding protein Golf alpha subunit. These findings suggest a role for this new hypothetical G-protein coupled receptor and for its still unknown ligand in the spleen and in the insulin-secreting beta cells.

[Spontaneous acquired localized neuropathies in childhood]. / Neuropatías localizadas adquiridas espontáneas en la infancia.

Acquired non-surgical, non-traumatic localized neuropathies, excluding cranial nerve disorders, are rare in infancy. We review the clinical histories of six children, studied for this disorders amongst a total of 2,105 children seen in the Neuropediatric Department of the Hospital Miguel Servet in Zaragoza. Two were diagnosed as familial neuropathy with pressure sensitive paralysis. Two plexopathies were considered to be familial brachial plexopathy with minor dysmorphic features. One case was diagnosed as idiopathic radial neuropathy and a further case as idiopathic lumbosacral plexopathy. We emphasize that although rare in pediatrics, spontaneous localized neuropathies often show constitutional pathology, frequently hereditary. Idiophatic cases may also be hereditary, and it may be difficult to confirm the diagnosis if there is no family history or phenotypic characteristics. Diagnosis depends on the personal and family history, physical examination, neurophysiological study, absence of abnormal neuroimaging findings and awareness of the possibility of this diagnosis. These disorders probably occur more often than is generally believed.

Regulated production of mature insulin by non-beta-cells.

Rat hepatoma cells were engineered to express, in a regulated manner, mature human insulin as an approach to the development of artificial beta-cells for insulin-dependent diabetes mellitus (IDDM) gene therapy. A chimeric gene obtained by linking a 2.4-kb fragment of the P-enolpyruvate carboxykinase (PEPCK) gene promoter to a human proinsulin gene (PEPCK/Insm), containing genetically engineered furin endoprotease cleavage sites, was stably transfected into FTO-2B rat hepatoma cells. The FTOInsm cells expressed high levels of insulin mRNA and protein after Northern blot or immunocytochemical analysis. High-performance liquid chromatography (HPLC) fractionation of culture medium and cell extracts revealed that about 90% of the proinsulin was processed to mature insulin. Insulin secretion was very fast, and 15 min after induction with dibutyryl cyclic AMP (Bt2cAMP) plus dexamethasone significant amounts of the hormone were released. Moreover, during the first hour, the rise in insulin concentration in the medium was 10-fold that detected in nontreated FTOInsm cells. Insulin produced by FTOInsm cells was biologically active because it blocked endogenous PEPCK gene expression and induced glucose uptake and lactate production. Thus, our results showed that genetically engineered FTOInsm hepatoma cells synthesized, processed, and secreted active insulin. The implantation of encapsulated engineered FTOInsm cells might provide a safe and practical therapeutic approach for IDDM treatment.

Effects of glucagon and glucagon-like peptide-1-(7-36) amide on C cells from rat thyroid and medullary thyroid carcinoma CA-77 cell line.

Glucagon is known to stimulate calcitonin secretion by thyroid C cells over a wide range of concentrations, raising the possibility of its interaction with several types of receptors. This study was designed to characterize receptors that mediate the effect of glucagon on a rat C cell line (CA-77). Binding studies, using radiolabeled [125I]glucagon and [125I]glucagon-like peptide-1-(7-36) amide ([125I]tGLP-1), to CA-77 plasma membranes demonstrated the presence of 1) a glucagon receptor with a dissociation constant (Kd) of 2.3 nM and relative potencies for structurally related peptides as follows: glucagon > oxyntomodulin > > tGLP-1; and 2) a tGLP-1 receptor with a Kd of 0.33 nM and relative potencies as follows: tGLP-1 > oxyntomodulin > glucagon. Glucagon stimulated calcitonin secretion from CA-77 cells in a dose-dependent manner over 4 orders of magnitude, with a maximal response of 312% over the basal value and an ED50 close to 50 nM. tGLP-1 induced a calcitonin release over 2 orders of magnitude, with a maximal response of 170% over the basal value and an ED50 close to 0.2 nM. Glucagon and tGLP-1 stimulated cAMP production in CA-77 cells to similar maximal levels over 4 and 2 orders of magnitude, respectively. The stimulation of cAMP production by glucagon at concentrations over 10 nM was suppressed by the tGLP-1 antagonist exendin-(9-39) amide, whereas the stimulation of calcitonin secretion was only partly abolished. Using a perifusion system of rat thyroid, glucagon and tGLP-1 stimulated calcitonin secretion in a calcium-dependent manner. It is concluded that glucagon and tGLP-1 receptors are expressed in the rat C cell line (CA-77) and in the normal rat thyroid. The effects of glucagon on calcitonin secretion observed at high concentrations are mediated in part through interaction with tGLP-1 receptors and via an additional non-cAMP-mediated mechanism.

Comparative effects of GLP-1-(7-36) amide, oxyntomodulin and glucagon on rabbit gastric parietal cell function.

We have investigated in vitro, the effects of glucagon-like peptide-1-(7-36) amide (GLP-1-(7-36) amide), oxyntomodulin and glucagon on two rabbit parietal cell-enriched fractions (F3, F3n), with parietal cell contents of 60% and 88%, respectively. Histamine (10(-5) M) stimulated [14C]aminopyrine accumulation to an amount of 850% in excess of the basal level, whereas GLP-1-(7-36) amide (10(-7) M) and oxyntomodulin (10(-6) M) induced increases of 50% and 30%, respectively. With a histamine concentration of 10(-6) M, [14C]aminopyrine accumulation was stimulated to 498% in excess of the basal level; GLP-1-(7-36) amide (10(-7) M) and oxyntomodulin (10(-7) M) induced increases of 18% and 15%, respectively. With these parameters, oxyntomodulin[19-37] and glucagon were without effect. Specific binding of [125I]GLP-1-(7-36) amide to parietal cell plasma membranes was inhibited dose-dependently by GLP-1-(7-36) amide, oxyntomodulin and glucagon with inhibitory concentrations of 0.25 nM, 65 nM and 800 nM, respectively. No specific binding of [125I]oxyntomodulin or [125I]glucagon was detectable. GLP-1-(7-36) amide receptor mRNA was only detected in parietal cell-enriched fractions. GLP-1-(7-36) amide, oxyntomodulin and glucagon stimulated parietal cell cAMP production to similar maximal levels with median values close to 0.28 nM, 10.5 nM and 331.7 nM, whereas oxyntomodulin[19-37] had no effect. The maximal cAMP production induced by GLP-1-(7-36) amide, oxyntomodulin or glucagon was additive to that induced by histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon-like peptide-1-(7-36) amide, oxyntomodulin, and glucagon interact with a common receptor in a somatostatin-secreting cell line.

Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3. tGLP-1, OXM, and glucagon stimulated somatostatin release and cAMP accumulation in RIN T3 cells to similar maximum levels, with ED50 values close to 0.2, 2, and 50 nM and 0.02, 0.3, and 8 nM, respectively. Binding of [125I]tGLP-1, [125I]OXM, and [125I]glucagon to RIN T3 plasma membranes was inhibited by the three peptides, with relative potencies as follows: tGLP-1 > OXM > glucagon. Whatever the tracer used, the IC50 for tGLP-1 was close to 0.15 nM and was shifted rightward for OXM and glucagon by about 1 and 2-3 orders of magnitude, respectively. Scatchard analyses for the three peptides were compatible with a single class of receptor sites displaying a similar maximal binding close to 2 pmol/mg protein. In the hamster lung fibroblast cell line CCL39 transfected with the receptor for tGLP-1, binding of [125I]tGLP-1 was inhibited by tGLP-1, OXM, and glucagon, with relative potencies close to those obtained with RIN T3 membranes. Chemical cross-linking of [125I]tGLP-1, [125I]OXM, and [125I]glucagon revealed a single band at 63,000 mol wt, the intensity of which was dose-dependently reduced by all three peptides. These data suggest that in the somatostatin-secreting cell line RIN T3, OXM and glucagon stimulate somatostatin release through a tGLP-1-preferring receptor. This suggests that some biological effects, previously described for these peptides, might be due to their interaction with this receptor.

Characterization of binding sites for oxyntomodulin on a somatostatin-secreting cell line (RIN T3).

Oxyntomodulin (OXM), a glucagon-containing peptide extended at its C-terminal end by an octapeptide, is a potent inhibitor of gastric acid secretion in rat and man. OXM appears to act on gastric mucosa at least partially through a stimulation of gastric somatostatin release. We have investigated the effects of OXM on a somatostatin-secreting cell line (RIN T3) derived from a radiation-induced rat insulinoma and characterized specific binding sites for this peptide. OXM increased somatostatin release with an ED50 of 2.3 nM. OXM also stimulated the cAMP accumulation in intact RIN T3 cells and adenylate cyclase activity in RIN T3 cell membranes with ED50 values of 0.5 and 11 nM, respectively. On these parameters, glucagon was 10-30 times less potent than OXM. Forskolin, isobutylmethylxanthine, and 8-bromo-cAMP mimicked the effect of OXM on somatostatin release. Specific binding for mono-[125I]OXM was dependent upon time and membrane concentration. Binding of mono-[125I]OXM was inhibited by OXM and glucagon in a concentration-dependent manner, with dissociation constants (Kd) of 4.5 and 43 nM, respectively. The nonhydrolyzable analogs of GTP (guanosine 5',3-O-(thio)triphosphate and guanosine 5' (beta,gamma-imino)triphosphate decreased the binding of mono-[125I]OXM to its binding sites. Covalent cross-linking of mono-[125I]OXM or mono-[125I]glucagon to RIN T3 cell membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single radiolabeled band at 63,000 mol wt, which differed from that observed after cross-linking with liver plasma membranes (55,000 mol wt). These results demonstrate the presence of specific high affinity binding sites for OXM in a somatostatin-secreting cell line (RIN T3) and their coupling to adenylate cyclase via guanine nucleotide-binding proteins.