Eosinophilic esophagitis: Pathophysiology, diagnosis, and management.

Eosinophilic esophagitis (EoE) is a multifactorial esophageal inflammation, with a genetic predisposition, which combines a deficient esophageal mucosal barrier, an abnormal immune reaction to environmental allergens mediated by Th2 interleukins, immediate esophageal lesions and dysmotility, with secondary remodeling and fibrosis. Symptoms include reflux, abdominal pain, and food impaction, with a variation according to age. Fibroscopy shows major and minor endoscopic and histologic criteria, with a mucosal count≥15 eosinophils/high power field (Eo/hpf). A new entity has been defined, where gastroesophageal reflux disease (GERD) and EoE share responsibility: the PPIs-sensitive form of EoE (PPI-REE). Children with fibroscopy showing≥15 Eo/hpf need a second endoscopy following 8 weeks of PPI treatment. EoE has a strong association with other atopic disorders. Allergy testing (specific IgE blood test and skin prick tests [SPTs]) identifies patients at risk of anaphylaxis (14.8% of cases). The dietary therapy is based on a 4- to 12-week elimination test followed by endoscopy to check the disappearance of eosinophilic infiltration. The "dietary approaches are the amino acid-based formula, the allergy testing-based targeted diet, and the six-food elimination diet (empirical elimination of milk, wheat, soy, eggs, peanut/nuts, and fish/seafood). A recent first-line trial elimination of milk has been suggested, with wheat as a second elimination, if necessary. Dietary therapy allows remission and catch-up growth in 65% of cases. Swallowed topical steroids (budesonide in viscous gel or fluticasone propionate for nebulization) are an alternative, for which efficacy varies according to clinical and/or histological criteria and with relapses occurring at dosage tapering. Their use may be restricted by side effects, such as oral and/or esophageal candidiasis. The impact on long-term bone health and growth is unknown. Maintenance therapy is not standardized and is team-dependent, combining or not elimination diets and long-term steroids. The long-term risk of EoE is esophageal stenosis (25%) and endoscopic dilation may be repeated. Biotherapies have shown isolated histological improvement without significant clinical efficacy.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Death switch for gene therapy: application to erythropoietin transgene expression.

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

CO adsorption on CoMo and NiMo sulfide catalysts: a combined IR and DFT study.

Experimental IR spectra of carbon monoxide adsorbed on a series of Mo/Al2O3, CoMo/Al2O3, and NiMo/Al2O3 sulfided catalysts have been compared to ab initio DFT calculations of CO adsorption on CoMo and NiMo model surfaces. This approach allows the main IR features of CO adsorbed on the sulfide phase to be assigned with an uncertainty of 15 cm(-1). On the CoMo system, the band at 2070 cm(-1) is specific of the promotion by Co and is assigned to CO interacting either with a Co atom or with a Mo atom adjacent to a Co atom. On the NiMo system, CO adsorption on Ni centers of the promoted phase leads to a high-wavenumber band at approximately 2120 cm(-1) that strongly overlaps the band at 2110 cm(-1) characteristic of nonpromoted Mo sites. For NiMo and CoMo catalysts, broad shoulders at low wave numbers (below 2060 cm(-1)) are characteristic of Mo centers adjacent to promoter atoms, indicating a partial decoration of the MoS2 edges by the promoter.

A new experimental cell for in situ and operandoX-ray absorption measurements in heterogeneous catalysis.

A new X-ray absorption cell dedicated to in situ and operando experiments in heterogeneous catalysis has been built and tested. The cell consists of several boron nitride and stainless steel plates linked together using graphite seals. It allows the measurement of XANES and EXAFS spectra of heterogeneous catalysts within a wide range of photon energies in transmission mode under the flow of various oxidative and reductive gas mixtures at elevated temperatures. The cell is compact and easy to build. Catalysts are loaded into the cell as powders. The use of boron nitride and a small beam pathlength in the cell result in a low absorption of the X-ray beam at lower energies. The cell was tested by in situ characterizing cobalt species during oxidative and reductive pre-treatments of a silica-supported Fischer-Tropsch catalyst. An operando study of methanol conversion over alumina-supported molybdenum catalysts was also carried out.

Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Oxygen tension and a pharmacological switch in the regulation of transgene expression for gene therapy.

BACKGROUND: The combination of physiologically and pharmacologically controlled elements may provide a means to ensure both the regulation and the safety of transgene expression--two major goals in gene therapy. METHODS: A two-gene modulation system was developed that uses the following three levels of control: (i) the hypoxia-responsive element directing the transcription of the tetracycline-controlled transactivator (tTA); (ii) part of the oxygen-degradation domain limiting the production of tTA in normoxia; and (iii) the tetracycline switch of the transactivator activity (the tet-off system). RESULTS: This triple-control system allowed high expression of the gene of interest (luciferase or erythropoietin) by transfected cells upon hypoxia and low expression under normoxia or in the presence of tetracycline. This control of transgene expression was also obtained in mouse tumors. CONCLUSIONS: This multiple-control system is of interest for spatially restricting transgene expression into hypoxic tumors, and for finely adjusting the expression level of a therapeutic protein to the oxygen supply in medical applications such as neoangiogenesis or the erythropoietin-mediated treatment of anemia.

Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo.

High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34(+) human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in beta-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in beta-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin. (Blood. 2001;97:3776-3782)

Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA.

OBJECTIVE: A new intramuscular DNA electrotransfer method for erythropoietin (EPO) expression was evaluated in the natural mouse model of human beta-thalassemia (Hbb-thal1) in terms of its ability to reverse the anemia and improve the thalassemic features of erythrocytes. MATERIALS AND METHODS: Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used. RESULTS: This procedure induced very high hematocrit levels in beta-thalassemic mice compared to nonelectrotransferred mice. The hematocrit increase was dose dependent, still increased 4 months after injection of plasmid DNA, and associated with a high transgenic EPO blood level in all mice (up to 2500 mU/mL of plasma). EPO gene electrotransfer not only led to a long-lasting and dose-dependent increase in the hematocrit but also to a 100% increase in the lifespan of erythrocytes of thalassemic mice. This was related to a nearly complete reestablishment of alpha/beta globin chain balance, as demonstrated by a marked decrease in unpaired alpha globin chain. Eight months after the first electrotransfer of pCMV-mEPO plasmid, reinjection of the same construct raised the hematocrit to a level close to that observed following the first electrotransfer. CONCLUSION: This is the first description of the use of plasmid DNA to achieve long-term improvement in a mouse model of a human genetic disorder.

Modulation of transduced erythropoietin expression by iron.

OBJECTIVE: Future prospects for gene therapy of chronic anemias involve expression of the erythropoietin transgene, which is regulated by oxygen tension. However, other factors such as cytokines or the iron load of erythropoietin-expressing cells can concomitantly modulate transgene expression, as shown for the expression of the endogenous erythropoietin gene in human cell lines and in animals. We tested the effects of iron overload or depletion on the expression of the mouse erythropoietin transgene (cDNA), driven by the hypoxia-regulated phosphoglycerate kinase 1 promoter. MATERIALS AND METHODS: Retrovirally transduced mouse cells (C3H fibroblasts or C2C12 myoblasts) were cultured in normoxia (room air, O2: 21%) or hypoxia (O2: 1.5%) in the presence or absence of hemin (an iron donor) or deferiprone (an iron chelator), both of which easily enter the cell. RESULTS: Hemin inhibited the hypoxia-induced expression of the transgene. In contrast, deferiprone enhanced the hypoxia-induced expression of the erythropoietin transgene and induced its expression in normoxia. CONCLUSION: These results show that, in addition to oxygen partial pressure, the intracellular iron content is critical in the modulation of hypoxia-regulated erythropoietin transgene expression.

Improvement of mouse beta-thalassemia upon erythropoietin delivery by encapsulated myoblasts.

The goal of the present study was to analyze if sustained delivery of elevated doses of recombinant erythropoietin (Epo), by genetically modified and immunoprotected allogenic cells, was able to correct the chronic anemia, characteristic of a spontaneous mouse model of beta-thalassemia (Hbb thal 1). Mouse C2C12 myoblast cells were transfected with a plasmid containing the mouse Epo cDNA and a mutated dihydrofolate reductase (DHFR) gene for gene amplification upon administration of increasing doses of methotrexate. In order to immunoprotect the transplanted cells, the stably modified cells were loaded into polyethersulfone microporus hollow fibers which were implanted subcutaneously into Hbb thal 1 mice. An increase in hematocrit starting 2 weeks after implantation was associated with elevated blood levels of Epo and an improved red blood cell phenotype. The latter indicated an improvement of cell morphology and membrane defects, in particular a reduced amount of free alpha hemoglobin chain, the hallmark of globin chain imbalance in beta-thalassemia. A reduction of reticulocyte count contrasting with the increase in hematocrit was also observed suggesting an improved erythrocyte survival. We conclude that the phenotype can be durably improved in some beta-thalassemic mice upon in vivo delivery of recombinant Epo by polymer encapsulated cells. Sustained elevated delivery of recombinant Epo holds promise for the treatment of beta-thalassemia-associated chronic anemia.

The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains.

Murine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Krüppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abundant in the developing central nervous system, and in the adult mouse expression is restricted largely to testis and brain. Here we show that at the protein level ZFP-37 is detected readily in neurons of the adult central nervous system but hardly in testis. In brain ZFP-37 is associated with nucleoli and appears to contact heterochromatin. Mouse and human ZFP-37 have a basic histone H1-like linker domain, located between KRAB and zinc finger regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a structural protein of the neuronal nucleus which plays a role in the maintenance of specialized chromatin domains.

The ovine SOX2 gene: sequence, chromosomal localization and gonadal expression.

The SOX gene family consists of a large number of embryonically expressed genes capable of encoding putative transcription factors and related by a DNA-binding domain, the HMG-box. We cloned and characterized the ovine SOX2 transcript using the screening of a testis (12dpp) cDNA library with a probe containing the SRY-HMG-box and we performed 3'RACE experiments. The ovine SOX2 sequence is strongly conserved in comparison to the human, mouse and chicken homologues and is located on sheep chromosome 1q33. The SOX2 expression pattern in developing gonads is consistent with the hypothesis that this gene plays a role in the germ cell line.

Transcription of Y- and X-linked genes in preimplantation ovine embryos.

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed.

Critical cooling and warming rates to avoid ice crystallization in small pieces of mammalian organs permeated with cryoprotective agents.

Measurements were made by differential scanning calorimetry on small pieces of rabbit kidney permeated with 2, 3-butanediol containing mainly the levo- and dextro-isomers. The critical cooling rates necessary to vitrify the pieces of organ, and the corresponding critical warming rates which are required to avoid crystallization in the vitrified samples, were determined. The dynamic method used for these determinations is described. The glass-forming tendency and the stability of the amorphous state were both greater in the kidney tissue samples than in the bulk cryoprotective solution. This result is discussed in the context of the lowering of the freezing point of water in emulsions and the promotion of supercooling in hydrogels and porous materials. In corresponding experiments with rat hearts impregnated with 1,2-propanediol, only the critical warming rate was reduced.

Characterization of ovine SRY transcript and developmental expression of genes involved in sexual differentiation.

In mammals, the presence of SRY, the sex-determining gene located on the Y chromosome is required to induce the gonadal anlage to differentiate as a testis, whereas its absence leads to the development of an ovary. We report here the characterization by 5' and 3' RACE analysis of several SRY transcripts which are expressed in the ovine male developing gonads. These transcripts were not detected in any other fetal tissues and were expressed only in the genital portion of the urogenital ridge. The temporal profile of SRY expression analyzed by RT-PCR suggests that in the sheep fetus the role of SRY is not limited to initiating Sertoli cell differentiation as in mice. Indeed, SRY transcripts persist after the full differentiation of the testis. In addition to SRY, other genes are known to be involved in mammalian sex determination: Wilms' tumor gene WT-1, steroidogenic factor gene Ftz-F1 (SF-1) and anti-Müllerian hormone (AMH). We investigated the expression patterns of these genes by RT-PCR during fetal development in sheep gonads. Concerning WT-1 and SF-1, our results are consistent with those described in mice where the earliest expression was detected before the sexual differentiation in both sexes. In male, the ontogenesis of AMH transcription corresponds to the seminiferous cords formation (30 dpc). In female, we have observed the presence of SF-1 transcripts from the undifferentiated stage until birth. In addition, P450 aromatase expression is detected from 30 dpc and is correlated with the presence of 17-beta estradiol in sheep ovary. These data reveal significant differences between rodent and ruminant models concerning the sex-determining pathway.

[Urachal adenocarcinoma associated with urothelial tumor of the bladder. Report of a case]. / Association d'un adénocarcinome de l'ouraque et d'une tumeur urothéliale de vessie. A propos d'un cas.

The authors report a rare association of adenocarcinoma of the urachus and urothelial bladder carcinoma. Treatment consisted of cystoprostatectomy with removal of the urachus and cutaneous ureterostomy. A review of the literature indicates that adjuvant treatment has yet to be defined. This is a chance association whose prognosis depends on the invasive nature of the urothelial tumour.

[Traumatic rupture of the bladder. Apropos of 26 cases]. / Les ruptures traumatiques de la vessie. A propos de 26 cas.

26 patients were seen with ruptured bladders at Grenoble Hospital during the last 15 years. Pelvic fractures were present in 95% of the cases. Bladder injuries were extraperitoneal (58%), intraperitoneal (27%), intraperitoneal and extraperitoneal (15%). Patients were hospitalised in Urological Department (40%) or General Surgical and Intensive Care (60%). All patients with intraperitoneal bladder injury, 61% with extraperitoneal ruptures were treated by surgical repair. 4 patients died, secondary to visceral associated injuries.

Localization of ZNF164, ZNF146, GGTA1, SOX2, PRLR and EEF2 on homoeologous cattle, sheep and goat chromosomes by fluorescent in situ hybridization and comparison with the human gene map.

The six following genes: zinc finger proteins 164 (ZNF164) and 146 (ZNF146), alpha-galactosyltransferase 1 (GGTA1), SRY-related HMG-box 2 (SOX2), prolactin receptor (PRLR) and elongatin factor 2 (EEF2) have been localized by fluorescent in situ hybridization respectively on bovine and caprine chromosomes 17, 18, 11, 1, 20 and 7 and on sheep chromosomes 17, 14, 3, 1, 16, and 5. The comparison of the results with the localization of these genes in man (except for ZNF164) confirm the correspondences between human and bovine chromosomes established from heterologous chromosome painting data.