TH-MYCN tumors, but not tumor-derived cell lines, are adrenergic lineage, GD2+, and responsive to anti-GD2 antibody therapy.

Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

[Epidemiology of fibrosing interstitial lung diseases in the department of Haute Garonne]. / EPIDemio : épidémiologie des pneumopathies interstitielles diffuses (PID) fibrosantes en Haute-Garonne.

EPIDemio study is a multicenter, prospective and observational study. The objective is to estimate the prevalence and incidence of fibrosing interstitial lung diseases (ILDs) in the department of Haute Garonne (31) in France. Fifty-five pulmonologists from the Toulouse university hospital and 8 private establishments participated in this study. Two hundred and fifty-six cases of fibrosing ILDs were reported (gross overall prevalence: 22.8/100,000 and estimated 30.1/100,000. Idiopathic ILDs represent 55.8% of fibrosing ILDs ahead of systemic disease-related ILDs (24.6%) and ILDs associated with environmental exposure (13.3%). Idiopathic pulmonary fibrosis (IPF) represents 35.9% of fibrosing ILDs, which corresponds to a minimal prevalence of 8.2/100,000 and an estimated prevalence of 11.2/100,000. This study confirms epidemiological data collected in France and Europe.

Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.

AIMS/HYPOTHESIS: Autotaxin is a lysophospholipase D that is secreted by adipocytes and whose expression is substantially up-regulated in obese, diabetic db/db mice. The aim of the present study was to depict the physiopathological and cellular mechanisms involved in regulation of adipocyte autotaxin expression. METHODS: Autotaxin mRNAs were quantified in adipose tissue from db/db mice (obese and highly diabetic type 2), gold-thioglucose-treated (GTG) mice (highly obese and moderately diabetic type 2), high-fat diet-fed (HFD) mice (obese and moderately diabetic type 2), streptozotocin-treated mice (thin and diabetic type 1), and massively obese humans with glucose intolerance. RESULTS: When compared to non-obese controls, autotaxin expression in db/db mice was significantly increased, but not in GTG, HFD, or streptozotocin-treated mice. During db/db mice development, up-regulation of autotaxin occurred only 3 weeks after the emergence of hyperinsulinaemia, and simultaneously with the emergence of hyperglycaaemia. Adipocytes from db/db mice exhibited a stronger impairment of insulin-stimulated glucose uptake than non-obese and HFD-induced obese mice. Autotaxin expression was up-regulated by treatment with TNFalpha (insulin resistance-promoting cytokine), and down-regulated by rosiglitazone treatment (insulin-sensitising compound) in 3T3F442A adipocytes. Finally, adipose tissue autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. CONCLUSIONS/INTERPRETATION: The present work demonstrates the existence of a db/db-specific up-regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression.

Binding of prostaglandins to human PPARgamma: tool assessment and new natural ligands.

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.

Oral tacrolimus treatment of severe colitis in children.

OBJECTIVE: To evaluate the efficacy of oral tacrolimus as an induction agent in steroid-refractory severe colitis. STUDY DESIGN: Open-label, multicenter trial of oral tacrolimus in patients with severe colitis. Patients not responding to conventional therapy received tacrolimus, 0.1 mg/kg/dose given twice a day, and the dosage was adjusted to achieve blood levels between 10 and 15 ng/mL. Response was defined as improvement in a number of clinical parameters (including abdominal pain, diarrhea, rectal bleeding, and cessation of transfusions). Patients who responded by 14 days continued to receive tacrolimus, and 6-mercaptopurine or azathioprine was added as a steroid-sparing agent 4 to 6 weeks after the tacrolimus was instituted. RESULTS: Fourteen patients were enrolled in the study. One patient elected to withdraw after 48 hours. Of the 13 remaining, 9 (69%) responded and were discharged. Tacrolimus was continued for 2 to 3 months in the responders, except for 1 patient who was given tacrolimus for 11 months. After 1 year of follow-up, only 5 (38%) patients were receiving maintenance therapy; the other 4 responders had undergone colectomy. CONCLUSION: Although tacrolimus is effective induction therapy for severe ulcerative or Crohn's colitis, fewer than 50% of patients treated will successfully achieve a long-term remission.

Optimization of aerosol deposition by pressure support in children with cystic fibrosis: an experimental and clinical study.

Nebulized aerosols are commonly used to deliver drugs into the lungs of patients with cystic fibrosis (CF). The aim of this study was to assess the effectiveness of pressure-support (PS) ventilation in increasing aerosol deposition within the lungs of children with CF. An in vitro study demonstrated the feasibility of coupling a breath-actuated nebulizer to a PS device. An in vivo study was done with 18 children (ages 6 to 21 yr) with clinically stable CF, each of whom underwent both a standard and a PS-driven ventilation scan (control session and PS session, respectively). In addition, a perfusion scan was used to determine lung outlines and to construct a geometric model for quantifying aerosol deposition by radioactivity counting in MBq. Homogeneity of nebulization was evaluated from the four first-order moments of aerosol distribution in the peripheral and central lung regions. The time-activity nebulization curve was linear in all patients, with higher slopes during the PS than during the control session (0.43 +/- 0.07 [mean +/- SD] MBq/min and 0.32 +/- 0.23 MBq/min, respectively; p < 0.018). Quantitatively, aerosol deposition was about 30% greater after the PS session (4.4 +/- 2.7 MBq) than after the control session (3.4 +/- 2.1 MBq; p < 0.05). Similarly, deposition efficacy (as a percentage of nebulizer output) was significantly better during the PS session than during the control session (15.3 +/- 8.3% versus 11.5 +/- 5.7%, p < 0.05). No differences in the regional deposition pattern or in homogeneity of uptake were observed. In conclusion, our data show that driving the delivery of a nebulized aerosol by noninvasive PS ventilation enhances total lung aerosol deposition without increasing particle impaction in the proximal airways.

Tumour suppressive properties of fibroblast growth factor receptor 2-IIIb in human bladder cancer.

FGFRs (fibroblast growth factor receptors) are encoded by four genes (FGFR1-4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5' region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identified in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which affected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.

A zinc chelator inhibiting gelatinases exerts potent in vitro anti-invasive effects.

Matrix metalloproteinases are zinc metalloenzymes involved in remodelling of the extracellular matrix. We compared the anti-invasive properties of a zinc ejector matrix metalloproteinase inhibitor with those of reference compounds (hydroxamic acid-based BB-94 and Ro-31-9790) which form inactive ternary complexes with the enzymes and the catalytic zinc. We show that the compound undecadenedioic acid bis-[[2-(3 H-imidazol-4-yl)-ethyl]-amide] (S 30372) is active against gelatinases, chelates zinc and exhibits enzymatic features compatible with the potential to extract zinc from gelatinases. We then used five invasive cell lines in the Matrigel invasion chamber assay (NIH-3T3 fibroblasts, Lewis lung carcinoma cells, EJ138 and J82 bladder carcinoma and HT1080 fibrosarcoma cells). With the exception of J82 cells which were unaffected by the three inhibitors, all remaining cells were substantially more sensitive to S 30372 in terms of maximal inhibition of invasion attained. This suggests that matrix metalloproteinase inhibitors with zinc chelating/ejecting properties may be more efficient in preventing tumor progression.

Balloon pulmonary valvuloplasty and stent implantation. For peripheral pulmonary artery stenosis in Alagille syndrome.

We report the case of a patient with Alagille syndrome and severe pulmonary valve and bilateral pulmonary artery branch stenosis. In this patient, transcatheter balloon pulmonary valvuloplasty combined with bilateral pulmonary artery angioplasty and stent placement provided excellent immediate results and long-term improvement.

Identifying and recruiting healthy control subjects from a managed care organization: a methodology for molecular epidemiological case-control studies of cancer.

Case-control studies with stringent matching criteria require large pools of healthy subjects from which to select matched controls. This paper describes a successful method of identifying a large pool of potential control subjects to participate in two molecular epidemiological case-control studies of lung cancer, each enrolling 400 case subjects and 400 control subjects. These studies are not population based, and the study base is not well-defined. Therefore, potential control subjects are being identified and recruited through 20 area clinic sites of a large multispecialty health maintenance organization. Because the research focus is driven by genetic hypotheses and we are controlling for multiple smoking-related variables, representativeness is of lesser concern. To identify potential control subjects, a one-page questionnaire is distributed to patients in the waiting room to assess contact information as well as data relevant to the case-control matching process. An average of 2,228 questionnaires are returned monthly toward a target pool of 40,000; of these, 59% of the respondents fulfill eligibility criteria as a control subject for one of the studies and are not averse to being contacted in the future for the purpose of research. When compared to former smokers and never smokers, current smokers in the control population were least likely to refuse further contact. A collaborative arrangement with a managed care organization offers a feasible mechanism through which researchers can access a large, ethnically diverse population of potential control subjects.

Selection of a histidine-containing inhibitor of gelatinases through deconvolution of combinatorial tetrapeptide libraries.

A fully automated peptide synthesizer was used to generate tetrapeptide sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their linear range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine-epsilon-amino caproic acid-beta-alanine-histidine (His-epsilon Ahx-beta Ala-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibition by selected tetrapeptides was also evaluated with two other techniques, a native type IV collagen degradation assay and a fluorogenic enzymatic assay, confirming the tetrapeptide potency. The His-epsilon Ahx-beta Ala-His tetrapeptide also inhibited purified human MMP-2 and MMP-9 and the corresponding enzymes present in conditioned media from human tumour cells. Finally, the length of the spacer between the two terminal histidines was found to be crucial to the inhibitory potential. This approach may thus be considered as a-successful strategy to yield specific peptide or pseudopeptide inhibitors, although their potency remains moderate, since it was measured before any chemical optimization was undertaken.

In vivo hippocampal (31)P NMR metabolites in Alzheimer's disease and ageing.

Memory loss is the most common early symptom of Alzheimer's disease (AD). For this study, we chose the hippocampi as regions of interest. The hippocampus, which is closely associated with memory processing, is known to be vulnerable to damage in the early stage of AD. We considered both inter-group (patients vs controls) and intra-group (right vs left hippocampus) comparisons. We examined seven patients meeting the DSM-III-R criteria of senile dementia and the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS - ADRDA) criteria of probable AD, and II aged controls. This study focused on the measurement of phosphorus 31 ((31)P) Nuclear Magnetic Resonance (NMR) spectroscopy metabolites in each hippocampus. We found significant differences in phosphorus metabolites for both intra-group comparison (pH shifted towards relative alkalosis in the left hippocampus of patients) and inter-group consideration (reduced phosphodiesters [Pde]and elevated gamma adenosine triphosphate (ATP) in the right hippocampus, higher inorganic phosphate (pHi) in the left hippocampus for patients as compared to controls). We suggest energy failure and membrane functional breakdown in patients compared to aged controls.

Mechanisms of growth retardation, drug therapy, and nutritional support in pediatric inflammatory bowel disease: a workshop sponsored by the north american and European societies for pediatric gastroenterology and nutrition.

SUMMARY: : Growth retardation is common in children with inflammatory bowel disease (IBD). The most sensitive measure of impaired growth, growth velocity, is abnormal in 65% of children with Crohn's disease. With treatment, growth velocity may return to normal, but catch-up growth is often incomplete and ultimate height lower than predicted. In some patients delayed puberty may compensate for poor growth earlier in life, and there is good evidence that even after menarche, significant growth can occur. Surgery may have a favorable impact on growth in the short term, but final height often remains reduced. The mechanism for growth failure in IBD is thought to be related to both prolonged periods of suboptimal nutritional intake and persistent inflammation. Growth throughout childhood is dependent on growth hormone and insulin-like growth factors (IGF). At puberty, androgens and estrogens also play a significant role in normal growth. Both malnutrition and inflammatory bowel disease result in low levels of IGF-1. With recovery, levels return to normal. Although little work has been done to measure the effects of chronic maintenance drug therapy on growth, it is known that children taking alternate-day prednisone grow normally and can exhibit catch-up growth. Nutritional therapy with tube feeding of elemental diets also improves nutrition and decreases inflammation. Children with either small-bowel Crohn's or Crohn's ileocolitis respond to tube feedings of both elemental or semielemental diets.

Substrate phosphorylation capacities of the major tyrosine protein kinase from the human promyelocytic cell line, HL-60.

The major tyrosine protein kinase, HPK40, isolated from HL-60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503-514), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid-treated enolase, actin and tubulin but not calmodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the 'src' family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corresponding peptide RRsrc, RRLIED-NEYTARG. VALYDYESR from the SH3 domain of pp60c-src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H-ras1) were examined as substrates. The presence of one or several acidic residues on the N alpha-side of tyrosine residue was identified as the only apparently favorable determinant.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraductal tetracycline therapy for the treatment of chronic recurrent parotitis.

Chronic recurrent parotitis (CRP) is recurrent parotid inflammation with non-obstructive sialectasis. Therapies which produce acinar atrophy or remove the acini are effective in treating CRP. Parotidectomy, tympanic neurectomy, duct ligation, and radiation therapy have either a low success rate or a high risk of morbidity. Intraductal antibiotic instillation has been proposed as a possible method of treatment. We hypothesized that the cytotoxic effects of tetracycline could produce acinar atrophy. A double-blind experiment of intraductal tetracycline instillation was performed in ten rabbits. Acinar atrophy and acute inflammation were found in 40% of the tetracycline treated glands; controls had a complete absence of these histologic changes. These results support the use of tetracycline instillation to produce acinar atrophy and therefore, intraductal tetracycline may be an effective, low-risk therapy for CRP. The clinical features of CRP will be reviewed and therapeutic implications discussed.

Limy bile syndrome.

Limy bile syndrome (LBS) is a rare condition in which a radiopaque gallbladder and/or bile ducts are noted on plain roentgenograms. LBS is caused by calcium carbonate precipitation in the bile and is usually associated with distal biliary tract obstruction. The etiology of limy bile syndrome is unclear; however, it may be a long-term complication of total parenteral nutrition.

Integrated system for the screening of the specificity of protein kinase inhibitors.

Tyrosine protein kinases (TPKs) play a major role in the transformation of cells. They are currently used as molecular targets for new generations of anticancer compounds. Numerous TPKs have been described from various tissues using either classical molecular biochemical techniques or cloning strategies. As a natural extension of these discoveries, a large number of "specific" inhibitors have been described in the literature. The major problem with these inhibitors is that there is no simple way to compare their specificity and/or selectivity from one report to another. We have set up a simple, straightforward technique to compare the inhibitory potency of 14 classical inhibitors towards six well-described and at least partially purified protein kinases. This technique is based on a new assay, easy to carry out and non-restrictive in terms of the type of protein substrate used. It permits direct comparisons between the results obtained from various sources. Data obtained showed that, when assessed in this integrated system, specificity and selectivity of many "classical" inhibitors are often weak, thus demonstrating that a universal technique such as ours is essential for the molecular screening of new protein kinase inhibitors. Compounds showing specificity for this panel of protein kinases will be more easily targeted to some defined types of oncogene and of transformed cells.

Myristoyl-CoA:protein N-myristoyltransferase activity in cancer cells. Purification and characterization of a cytosolic isoform from the murine leukemia cell line L1210.

Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the N-terminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) Eur. J. Biochem. 201, 257-263], a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with [14C]myristoyl-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC50 of about 0.1 microM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.