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In 2016, the WHO included haemoglobin values within normal ranges as a diagnostic criterion for Polycythaemia Vera (PV). Since then, concerns have arisen that a large number of patients are undergoing unnecessary screening for PV. To address this issue, we estimated the prevalence of JAK2 p.V617F in individuals with elevated haemoglobin or haematocrit and developed and validated a screening algorithm for PV. A total of 15,366 blood counts performed in seven non-consecutive days were reviewed, of which 1001 were selected for subsequent JAK2 p.V617F mutation screening. Eight (0.8%) new JAK2 p.V617F-mutated cases were detected. From ROC curves, a two-step algorithm was developed based on the optimal cut-off for the detection of the JAK2 p.V617F mutation. The algorithm was prospectively validated in an independent cohort of 15,298 blood counts. A total of 1595 (10.4%) cases met the criterion for haemoglobin or haematocrit, of whom 581 passed to step 2 (3.8% of the total). The JAK2 p.V617F mutation was detected in 7 of the 501 patients tested, which accounts for 0.04% of the total cohort and 0.4% of patients with erythrocytosis. In conclusion, this data show that our two-step algorithm improves the selection of candidates for JAK2 p.V617F testing.

Sol-gel derived nano-glass-ceramics with composition 95SiO2-5BaY0.78-xGdxYb0.2Tm0.02F5, where x = 0, 0.2, 0.4, 0.6 and 0.78, were obtained after adequate thermal treatments of precursor glasses. X-ray diffraction, transmission electron microscope and energy dispersive X-ray spectroscopy, confirmed the precipitation and distribution of nanocrystals, with sizes around 10–13 nm, in an amorphous silica network. Under excitation at 980 nm, along with blue, red and NIR characteristic up-conversion emissions of Tm3+ ions, intense UV up-conversion emissions coming from 1I6 and 1D2 levels of Tm3+ ions were also observed in all the studied samples. Additionally, when substituting Y3+ by Gd3+ ions, UV up-conversion emissions coming from Gd3+ ions, along with a reduction of the UV up-conversion emissions of Tm3+ ions, were observed, involving an energy transfer process from Yb3+/Tm3+ systems to Gd3+ ions. UC mechanisms and energy transfer processes were confirmed by transient emission and pump power measurements. Moreover, power dependence analysis revealed saturation effects in all up-conversion emissions, even at low pump power. Results suggest that these highly efficient up-conversion nano-glass-ceramics present potential applications as UV-blue solid state laser materials and solid state lighting.

PRPS1 encodes isoform I of phosphoribosylpyrophosphate synthetase (PRS-I), a key enzyme in nucleotide biosynthesis. Different missense mutations in PRPS1 cause a variety of disorders that include PRS-I superactivity, nonsyndromic sensorineural hearing impairment, Charcot-Marie-Tooth disease, and Arts syndrome. It has been proposed that each mutation would result in a specific phenotype, depending on its effects on the structure and function of the enzyme.Thirteen Spanish unrelated families segregating X-linked hearing impairment were screened for PRPS1 mutations by Sanger sequencing. In two positive pedigrees, segregation of mutations was studied, and clinical data from affected subjects were compared.We report two novel missense mutations in PRPS1, p.Ile275Thr and p.Gly306Glu, which were found in the propositi of two unrelated Spanish families, both subjects presenting with nonsyndromic hearing impairment. Further investigation revealed syndromic features in other hemizygous carriers from one of the pedigrees. Sequencing of genes that are functionally related to PRPS1 did not reveal any candidate variant that might act as a phenotype modifier.This case of intrafamilial phenotypic variation associated with a single PRPS1 mutation complicates the genotype-phenotype correlations, which makes genetic counseling of mutation carriers difficult because of the wide spectrum of severity of the associated disorders.

The inner ear is a very complex sensory organ whose development and function depend on finely balanced interactions among diverse cell types. The many different kinds of inner ear supporting cells play the essential roles of providing physical and physiological support to sensory hair cells and of maintaining cochlear homeostasis. Appropriately enough, the gene most commonly mutated among subjects with hereditary hearing impairment (HI), GJB2, encodes the connexin-26 (Cx26) gap-junction channel protein that underlies both intercellular communication among supporting cells and homeostasis of the cochlear fluids, endolymph and perilymph. GJB2 lies at the DFNB1 locus on 13q12. The specific kind of HI associated with this locus is caused by recessively-inherited mutations that inactivate the two alleles of the GJB2 gene, either in homozygous or compound heterozygous states. We describe the many diverse classes of genetic alterations that result in DFNB1 HI, such as large deletions that either destroy the GJB2 gene or remove a regulatory element essential for GJB2 expression, point mutations that interfere with promoter function or splicing, and small insertions or deletions and nucleotide substitutions that target the GJB2 coding sequence. We focus on how these alterations disrupt GJB2 and Cx26 functions and on their different effects on cochlear development and physiology. We finally discuss the diversity of clinical features of DFNB1 HI as regards severity, age of onset, inner ear malformations and vestibular dysfunction, highlighting the areas where future research should be concentrated.

Resumen: Introducción: Los pacientes críticos frecuentemente presentan inestabilidad hemodinámica que puede verse empeorada por la administración de algunos fármacos. El omeprazol es un fármaco muy empleado en la profilaxis de la hemorragia digestiva en estos pacientes, pero se desconocen sus efectos cardiovasculares. El objetivo fue estudiar los cambios hemodinámicos producidos por la administración de omeprazol por vía intravenosa en niños críticos y analizar si existen diferencias entre 2 dosis diferentes de omeprazol. Material y métodos: Se realizó un estudio prospectivo observacional aleatorizado, que incluyó niños críticos entre un mes y 14 años de edad, que precisaban profilaxis de hemorragia digestiva. La muestra aleatoria fue de 37 pacientes de los cuales, 19 recibieron omeprazol por vía intravenosa 0,5 mg/kg cada 12 h y 18 omeprazol por vía intravenosa 1 mg/kg cada 12 h. La dosis correspondiente se administró en 20 min mediante bomba de infusión continua. Se recogieron la frecuencia cardiaca, presión arterial sistólica, media y diastólica, presión venosa central y el electrocardiograma basal, a los 15, 30, 60 y 120 min de la infusión. Resultados: La edad media fue de 27,7 ± 33,3 meses. Todos los pacientes recibieron dopamina y 14, adrenalina. No se produjeron modificaciones significativas en ninguna de las variables estudiadas. Ningún paciente precisó modificación del tratamiento inotrópico. No se objetivaron diferencias entre las 2 dosis de omeprazol. Conclusiones: El omeprazol por vía intravenosa es un fármaco hemodinámicamente seguro en niños críticos, a cualquiera de las 2 dosis utilizadas. Abstract: Introduction: Critical patients usually have hemodynamic disturbances which may become worse by the administration of some drugs. Omeprazole is a drug used in the prophylaxis of the gastrointestinal bleeding in these patients, but its cardiovascular effects are unknown. The objective was to study the hemodynamic changes produced by intravenous omeprazole in critically ill children and to find out if there are differences between two different doses of omeprazole. Material and methods: A randomized prospective observational study was performed on 37 critically ill children aged from 1 month to 14 years of age who required prophylaxis for gastrointestinal bleeding. Of these, 19 received intravenous omeprazole 0.5 mg/kg every 12 hours, and 18 received intravenous omeprazole 1 mg/kg every 12 hours. Intravenous omeprazole was administered in 20 minutes by continuous infusion pump. Heart rate, systolic, diastolic and mean arterial blood pressure, central venous pressure and ECG were recorded at baseline, and at 15, 30, 60 and 120 minutes of the infusion. Results: There were no significant changes in the electrocardiogram, heart rate, blood pressure and central venous pressure. No patients required inotropic therapy modification. There were no differences between the two doses of omeprazole. Conclusions: Intravenous omeprazole administration of 0.5 mg/kg and 1 mg/kg is a hemodynamically safe drug in critically ill children.

Resumen: Objetivo: Valorar si un espirómetro puede medir el volumen corriente en ventilación de alta frecuencia oscilatoria (VAFO) y por jet (VAFJ) y analizar el efecto de los cambios en los parámetros del respirador. Métodos: Se realizó un estudio con pulmones de prueba pediátricos y en cerdos. Para la VAFO se utilizó el respirador Sensormedics 3100BR y para la VAFJ el respirador Paravent PateR. La medición del volumen corriente (VC) se realizó con un espirómetro D-Fend. Se realizaron cambios en la frecuencia, amplitud y presión media en la vía aérea (PMVA), registrándose los cambios en el VC y las presiones. Resultados: El espirómetro midió el VC en los pulmones de prueba y en los cerdos, pero no pudo medir VC menores de 8 ml, cuando la amplitud era mayor de 55 cmH2O o la PMVA mayor de 30 cmH2O. En la VAFO existió una correlación entre la amplitud y el VC y en la VAFJ entre la presión y el VC. En ambos respiradores existió una correlación negativa entre la frecuencia y el VC. Conclusiones: El espirómetro D-Fend puede medir el volumen corriente durante la VAFO y la VAFJ, pero no es capaz de medir VC menores de 8 o cuando se utilizan amplitudes o presiones elevadas. Abstract: Objective: To ascertain whether a spirometer can measure tidal volume (TV) during high frequency oscillatory ventilation (HFOV) and high frequency jet ventilation (HFJV), and to analyse the effect of changes in ventilator settings. Methods: The study was performed with paediatric porcine lung models submitted to HFOV with a Sensormedics 3100 ventilator and HFJV with a Paravent PateR ventilator connected to a D-Fend spirometer. Programmed frequency, amplitude, and mean airway pressure (MAP) were changed in the ventilator, and TV and pressures were recorded using the spirometer. Results: The spirometer measured TV in the paediatric lung models and piglets, but could not measure TV less than 8 ml, when the pressure amplitude was higher than 55 cmH2O or the MAP was higher than 30 cmH2O. With HFOV there was a correlation between amplitude and tidal volume, and a positive correlation between pressure and TV with HFJV. With both respirators there was a negative correlation between frequency and TV. Conclusions: The D-Fend spirometer can measure tidal volume and pressure during HFOV and HFJV. However, it does not work with volumes lower than 8 ml, and high amplitude or mean airway pressure.

Targeting of numerous transmembrane proteins to the cell surface is thought to depend on their recognition by cargo receptors that interact with the adaptor machinery for anterograde traffic at the distal end of the Golgi complex. We report here on consortin, a novel integral membrane protein that is predicted to be intrinsically disordered, i.e. that contains large segments whose native state is unstructured. We identified consortin as a binding partner of connexins, the building blocks of gap junctions. Consortin is located at the trans-Golgi network (TGN), in tubulovesicular transport organelles, and at the plasma membrane. It directly interacts with the TGN clathrin adaptors GGA1 and GGA2, and disruption of this interaction by expression of a consortin mutant lacking the acidic cluster-dileucine (DXXLL) GGA interaction motif causes an intracellular accumulation of several connexins. RNA interference-mediated silencing of consortin expression in HeLa cells blocks the cell surface targeting of these connexins, which accumulate intracellularly, whereas partial depletion and redistribution of the consortin pool slows down the intracellular degradation of gap junction plaques. Altogether, our results show that, by studying connexin trafficking, we have identified the first TGN cargo receptor for the targeting of transmembrane proteins to the plasma membrane. The identification of consortin provides in addition a potential target for therapies aimed at diseases in which connexin traffic is altered, including cardiac ischemia, peripheral neuropathies, cataracts and hearing impairment. Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_146105.

Auditory neuropathy is a particular type of hearing impairment in which neural transmission of the auditory signal is impaired, while cochlear outer hair cells remain functional. Here we report on DFNB59, a newly identified gene on chromosome 2q31.1-q31.3 mutated in four families segregating autosomal recessive auditory neuropathy. DFNB59 encodes pejvakin, a 352-residue protein. Pejvakin is a paralog of DFNA5, a protein of unknown function also involved in deafness. By immunohistofluorescence, pejvakin is detected in the cell bodies of neurons of the afferent auditory pathway. Furthermore, Dfnb59 knock-in mice, homozygous for the R183W variant identified in one DFNB59 family, show abnormal auditory brainstem responses indicative of neuronal dysfunction along the auditory pathway. Unlike previously described sensorineural deafness genes, all of which underlie cochlear cell pathologies, DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect.

Hearing impairment is a common and highly heterogeneous sensory disorder. Genetic causes are thought to be responsible for more than 60% of the cases in developed countries.1 In the majority of cases, non-syndromic hearing impairment is inherited in an autosomal recessive pattern.2 Thirty eight different loci and 20 genes for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been identified to date.3 In many populations, up to 50% of all cases of ARNSHI are caused by mutations in the DFNB1 locus (MIM 220290) on 13q12.4 This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26),5 which belongs to a family of transmembrane proteins with about 20 members in humans. Hexamers of connexins (connexons) are displayed in the plasma membrane. Docking of connexons on the surfaces of two adjacent cells results in the formation of intercellular gap junction channels.6 Several different connexins, including Cx26, have been shown to participate in the complex gap junction networks of the cochlea.7,8 It has been postulated that these networks play a key role in potassium homeostasis, which is essential for the sound transduction mechanism.9 Given the high prevalence of DFNB1 deafness, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non-syndromic hearing impairment of unknown cause.10 However, the finding of a large number of affected subjects with only one GJB2 mutant allele complicates the molecular diagnosis of DFNB1 deafness. In different studies, these have accounted for 10–50% of deaf subjects with GJB2 mutations.4 It was hypothesised that there could be other mutations in the DFNB1 locus but outside the GJB2 gene. This hypothesis gained support by the finding of a deletion in the DFNB1 locus outside GJB2 but truncating the neighbouring GJB6 gene (MIM 604418), which …

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

Inherited hearing impairment affects one in 2,000 newborns. Nonsyndromic prelingual forms are inherited mainly as autosomal recessive traits, for which 16 genes are currently known. Mutations in the genes encoding connexins 26 and 30 account for up to 50% of these cases. However, the individual contribution of the remaining genes to the whole remains undetermined. In addition, for most of the genes there is a need for studies on genotype–phenotype correlations, to identify distinctive clinical features which may direct the molecular diagnosis to specific genes. Here we present a mutation analysis and a genotype–phenotype correlation study on the gene encoding otoferlin (OTOF), responsible for the DFNB9 subtype of prelingual hearing impairment. Four novel mutations were identified: c.2122C>T (p.Arg708Ter), c.4275G>A (p.Trp1425Ter), c.4362+2T>G, and c.5860_5862delATC (p.Ile1954del). A total of 37 subjects with muta-tions in OTOF were studied clinically. They were phenotypically homogeneous, having profound hearing impairment with very early onset, as shown by pure-tone audiometry and auditory brainstem responses. Magnetic resonance imaging and computed tomography did not reveal any inner ear malformation. Unexpectedly, transient evoked otoacoustic emissions (TEOAEs) were present, either bilaterally or unilaterally in 11 subjects. Altogether, clinical data of these subjects met the diagnostic criteria of auditory neuropathy. A total of 10 subjects had been successfully provided with cochlear implants. The results of our study indicate that genetic diagnosis of subjects with auditory neuropathy and profound hearing impairment should be directed to the otoferlin gene. Our data are of concern to universal screening programs which use TEOAEs as the first detection test for hearing impairment in newborns, since this technique may overlook a nonnegligible proportion of cases. Hum Mutat 22:451–456, 2003. © 2003 Wiley-Liss, Inc.

Hearing impairment is the most prevalent sensory disorder and genetic causes are thought to be responsible for over 60% of the cases in developed countries.1 Inherited hearing impairment is highly heterogeneous from both the clinical and genetic points of view.1,2 It varies in age of onset, severity, and audiological characteristics, and it can be associated or not with other clinical features (syndromic or non-syndromic hearing impairment). Genetic transmission includes autosomal (dominant and recessive), X linked, and maternal inheritance patterns. This unparalleled heterogeneity is well illustrated by the fact that over 70 loci in the nuclear genome have been reported to be involved in non-syndromic hearing impairment, and about 30 genes have been isolated from their critical intervals.3 Furthermore, a number of different mutations in several genes of the mitochondrial genome are responsible for syndromic and non-syndromic forms of hearing loss.4,5 Mutations responsible for maternally inherited non-syndromic hearing loss are so far confined to only two genes in the mitochondrial genome. These include mutations 7510T>C6 and 7511T>C7 in the tRNASer(UCN) gene, and 1095T>C8 and 1555A>G9 in the gene for the 12S rRNA. This last mutation is responsible for a dual phenotype, since it also confers increased susceptibility to the ototoxic action of aminoglycoside antibiotics.9 Most of these mutations have been reported in a small number of families from several countries, with the exception of 1555A>G, which seems to be more frequent than the others,10–13 although its real prevalence remains to be determined in most populations. Remarkably, in Spain it accounts for about 15–20% of all familial cases of non-syndromic hearing loss, irrespective of their mode of inheritance and age of onset14,15 (our unpublished results). In a majority of these patients, the hearing loss is not …

Inherited hearing impairment affects about 1 in 2000 newborns. Up to 50 percent of all patients with autosomal recessive nonsyndromic prelingual deafness in different populations have mutations in the gene encoding the gap-junction protein connexin 26 (GJB2) at locus DFNB1 on chromosome 13q12. However, a large fraction (10 to 42 percent) of patients with GJB2 mutations have only one mutant allele; the accompanying mutation has not been identified. DFNB1-linked familial cases with no mutation in GJB2 have also been reported.We evaluated 33 unrelated probands with nonsyndromic prelingual deafness who had only one GJB2 mutant allele. Nine subjects had evidence of linkage to DFNB1. We used haplotype analysis for markers on 13q12 to search for mutations other than the one involving GJB2.We identified a 342-kb deletion in the gene encoding connexin 30 (GJB6), a protein that is reported to be expressed with connexin 26 in the inner ear. The deletion extended distally to GJB2, which remained intact. The break-point junction of the deletion was isolated and sequenced, and a specific diagnostic test was developed for this common mutation. Twenty-two of the 33 subjects were heterozygous for both the GJB6 and GJB2 mutations, including all 9 with evidence of linkage to DFNB1. Two subjects were homozygous for the GJB6 mutation.A 342-kb deletion in GJB6 is the second most frequent mutation causing prelingual deafness in the Spanish population. Our data suggest that mutations in the complex locus DFNB1, which contains two genes (GJB2 and GJB6), can result in a monogenic or a digenic pattern of inheritance of prelingual deafness.

PURPOSE The aim of this study was to deepen our knowledge on the basis of intrafamilial genetic heterogeneity of inherited retinal dystrophies (RD) to further discern the contribution of individual alleles to the pathology. METHODS Families with intrafamilial locus and/or allelic heterogeneity were selected from a cohort of 873 characterized of 2468 unrelated RD families. Clinical examination included visual field assessments, electrophysiology, fundus examination, and audiogram. Molecular characterization was performed using a combination of different methods: genotyping microarray, single strand conformational polymorphism (SSCP), denaturing high pressure liquid chromatography (dHPLC), high resolution melt (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, whole-genome homozygosity mapping, and next-generation sequencing (NGS). RESULTS Overall, intrafamilial genetic heterogeneity was encountered in a total of 8 pedigrees. There were 5 of 873 families (~0.6%) with causative mutations in more than one gene (locus heterogeneity), involving the genes: (1) USH2A, RDH12, and TULP1; (2) PDE6B and a new candidate gene; (3) CERKL and CRB1; (4) BBS1 and C2orf71; and (5) ABCA4 and CRB1. Typically, in these cases, each mutated gene was associated with different phenotypes. In the 3 other families (~0.35%), different mutations in the same gene (allelic heterogeneity) were found, including the frequent RD genes ABCA4 and CRB1. CONCLUSIONS This systematic research estimates that the frequency of overall mutation load promoting RD intrafamilial heterogeneity in our cohort of Spanish families is almost 1%. The identification of the genetic mechanisms underlying RD locus and allelic heterogeneity is essential to discriminate the real contribution of the monoallelic mutations to the disease, especially in the NGS era. Moreover, it is decisive to provide an accurate genetic counseling and in disease treatment.

The Escherichia coli K-12 sheA gene encodes a pore-forming hemolysin that is secreted to the medium by a hitherto unidentified mechanism. To study SheA secretion, we constructed fusions between SheA and the mature form of the periplasmic enzyme beta-lactamase, and performed site-directed mutagenesis on these constructs. The SheA-Bla and Bla-SheA hybrid proteins displayed hemolytic activity and were efficiently exported to the extracellular medium. Our results with mutant hybrid proteins show that secretion of SheA is independent of its cytolytic activity, that secretion is paralleled by a transient leakage of periplasmic contents to the extracellular medium, and that deletion of the 11 C-terminal residues of SheA has no effect on its secretion and cytolytic activity.

Several errors were introduced in the preparation of this article for publication. The word "connexin" has been incorrectly substituted by "connection" throughout the article.

Haemolytic toxins belong to one of several classes of virulence factors that contribute to bacterial pathogenicity. The non-pathogenic Escherichia coli K-12 laboratory strain was considered for years to be non-haemolytic. However, overproduction of several transcriptional regulators induced the appearance of a haemolytic activity that is absent under usual laboratory growth conditions. In this work, we have cloned and characterized an E. coli K-12 gene, sheA, whose overexpression results in a haemolytic phenotype. It maps to min 27 on the genetic map, and codes for a 34-kDa polypeptide with at least one putative transmembrane segment. This polypeptide, which has neither signal peptide nor other known secretory motifs, is secreted to the medium during the exponential growth phase. In vitro coupled transcription/translation assays, using a plasmid carrying only the sheA gene as template, resulted in the production of a polypeptide with haemolytic activity per se. Our results demonstrate that the sheA gene actually encodes the E. coli K-12 chromosomal haemolysin. The SheA haemolysin does not resemble other known cytolytic toxins, and it may represent the prototype of a novel family, as suggested by the presence of homologues in several E. coli pathogenic strains and in Shigella flexneri.

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices. In addition to the expected thrombocytosis after 7 to 10 days of daily injection of high doses of PEG-rHuMGDF, a transient decrease in peripheral red blood cell numbers and hemoglobin is noted accompanied in the bone marrow by megakaryocytic hyperplasia, myeloid hyperplasia, erythroid and lymphoid hypoplasia, and deposition of a fine network of reticulin fibers. Splenomegaly, an increase in splenic megakaryocytes, and extramedullary hematopoiesis accompany the hematologic changes in the peripheral blood and marrow to complete a spectrum of pathologic features similar to those reported in patients with myelofibrosis and megakaryocyte hyperplasia. However, all the PEG-rHuMGDF-initiated hematopathology including the increase in marrow reticulin is completely and rapidly reversible upon the cessation of administration of PEG-rHuMGDF. Thus, transient hyperplastic proliferation of megakaryocytes does not cause irreversible tissue injury. Furthermore, PEG-rHuMGDF completely ameliorates carboplatin-induced thrombocytopenia at a low-dose that does not cause the hematopathology associated with myelofibrosis.

Gjb2andGjb6,two contiguous genes respectively encoding the gap junction protein connexin26 (Cx26) and connexin 30 (Cx30) display overlapping expression in the inner ear. Both have been linked to the most frequent monogenic hearing impairment, the recessive isolated deafness DFNB1. Although there is robust evidence for the direct involvement of Cx26 in cochlear functions, the contribution of Cx30 is unclear since deletion ofCx30strongly downregulatesCx26both in human and in mouse. Thus, it is imperative that any role of Cx30 in audition be clearly evaluated. Here, we developed a newCx30knock-out mouse model (Cx30Δ/Δ) in which half ofCx26expression was preserved. Our results show thatCx30andCx26coordinated expression is dependent on the spacing of their surrounding chromosomic region, and thatCx30Δ/Δmutants display normal hearing. Thus, in deaf patients withGJB6deletion as well as in the previousCx30knock-out mouse model, defectiveCx26expression is the likely cause of deafness, and in contrast to current opinion, Cx30 is dispensable for cochlear functions.

The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connexin-26) [corrected] gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connexin-26 [corrected] encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects.

Megakaryocyte growth and development factor (MGDF) administered intraperitoneally (IP) to mice causes a dose-dependent thrombocytosis accompanied by a decrease in mean platelet volume. MGDF increases the number of megakaryocytes in the bone marrow and spleen. MGDF does not affect the circulating number of leukocytes. Carboplatin, a chemotherapeutic agent that causes thrombocytopenia in humans, administered to mice as a single IP injection at a nonlethal dose causes a significant, but reversible thrombocytopenia. The carboplatin- induced thrombocytopenia is accompanied by an increase in circulating endogenous MGDF that precedes the return of circulating platelets to a normal level. MGDF mRNA is constitutively present in the liver. After carboplatin treatment, hepatic MGDF mRNA does not increase in concordance with circulating MGDF. Circulating soluble MGDF receptor levels (c-mpl) do not change significantly during the course of carboplatin-induced thrombocytopenia. MGDF injected IP once daily beginning 1 day after injection of carboplatin reverses carboplatin- induced thrombocytopenia in a dose-dependent fashion. The normalization of circulating platelet numbers in carboplatin plus MGDF-treated mice is accompanied by a normalization of megakaryocyte numbers in the bone marrow. In conclusion, MGDF, by increasing the number of marrow megakaryocytes and circulating platelets is an effective therapy for carboplatin-induced thrombocytopenia in mice.

Item does not contain fulltext Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow "U" or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding alpha-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.

Lipopolysaccharide (LPS) injected into the trachea of rats was found to induce the secretion of leukemia inhibitory factor (LIF) into bronchoalveolar lavage (BAL) fluid with a maximum expression of LIF after 2-12 h. The acute pulmonary neutrophilic inflammation caused by the intratracheal injection of bacterial endotoxin (LPS) could be inhibited by the intratracheal coinjection of recombinant LIF. Compared with intratracheal injection of LPS alone, intratracheal coinjection of LIF and LPS decreases the number of BAL neutrophils obtained 6 h later by approximately 50% (P < 0.0001). LIF decreased the amount of the proinflammatory cytokine tumor necrosis factor (TNF), but not the amount of the anti-inflammatory cytokine interleukin (IL)-6, in the BAL fluid of LPS-injected rats. Similarly, intravenous LIF was found to decrease TNF expression, but increase IL-6 expression, in the serum of rats receiving intravenous LPS. Intravenous LIF, even in the absence of LPS, was found to cause IL-6 expression. In conclusion, intratracheal LPS initiates the secretion of endogenous LIF into the alveolar space where LIF may contribute to the downregulation of LPS-initiated acute neutrophilic inflammation by downregulating expression of TNF. LIF may down-regulate LPS-initiated TNF expression at least in part indirectly by upregulating expression of IL-6, a cytokine known to downregulate LPS-initiated TNF expression.

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

1. The articular capsule or ligament, of the primary spines of the sea-urchin Eucidaris tribuloides behaves as a classical excitable tissue, responding with a shortening or contracture to a variety of stimuli including the cholinergic agonists acetylcholine (ACh), methacholine (MeACh), carbamylcholine (CCh) and nicotine. 2. d-Tubocurarine failed to decrease the amplitude of the acetylcholine-induced contractures, while the contractures elicited by methacholine were blocked by atropine. Neostigmine, but not eserine, increased the amplitude of ACh-induced contractures, suggesting the presence of AChE in the preparation. 3. The cholinergic agonists induced contracture of the ligament, but had quite different kinetics, the rate of rise of the contracture being fastest for ACh and decreasing in the following order: CCh, MeACh and nicotine. 4. Tyramine and octopamine exert an inhibitory action on "catch" and a relaxing effect on the contracting ligament. 5. The time courses of the contractures elicited in the same preparation were virtually identical to those of "catch". Therefore, we propose that "catch" and contracture are only two different aspects of the same phenomenon; namely the contracture of the muscle fibers present in the ligament.

1. This paper describes the effects of several cholinergic agonists and antagonists, and of β-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides. 2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely. 3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M3 muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine. 4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules. 5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of aeetyl-cholinesterase in the ligament. 6. β-Phenylethy lamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament. 7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.

We studied the effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.

The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.

Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF- induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal- type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells.

Macrophage colony-stimulating factor (recombinant human M-CSF) given as a single intravenous injection to Lewis rats induces a dose-dependent peripheral monocytosis, neutrophilia, and lymphopenia. The monocytosis peaks at 28 to 32 hours with a seven- to eightfold increase in the number of circulating monocytes and promonocytes. The peripheral monocytosis is accompanied by a slight increase in marrow blasts, promonocytes, and monocytes. A monocytopenia reaching a nadir at 15 minutes precedes the monocytosis, suggesting that M-CSF activates circulating monocytes and causes intravascular margination. The M-CSF- induced neutrophilia and lymphopenia are relatively mild in magnitude, are observed between 2 and 16 hours after injection, and are no longer evident at later time-points. The monocytosis was at least partially inhibited by dexamethasone. M-CSF-induced monocytosis most likely reflects a direct effect of M-CSF on marrow monocyte precursor proliferation, maturation, and release, whereas the neutrophilia and lymphopenia may reflect indirect effects mediated by the known ability of M-CSF to cause the release of other cytokines.

Multi-colony stimulating factor (Multi-CSF, interleukin-3, IL-3) and granulocyte-CSF (G-CSF) administered concurrently as an intravenous (IV) injection induce a peripheral neutrophilia that is approximately additive in comparison with the neutrophilia induced by IL-3 and G-CSF individually. The bone marrow (BM) at 12 hours is depleted of mature neutrophils and shows a left-shifted myeloid hyperplasia, consistent with the neutrophil-releasing and myeloproliferative activities of both IL-3 and G-CSF individually. The BM at 24 hours shows a replenished reserve of mature neutrophils and a synergistic left-shifted myeloid hyperplasia as compared with IL-3 and G-CSF alone. Daily IV injections of IL-3 plus G-CSF for 1 week also induce an approximately additive daily peripheral neutrophilia. The BM after a week's administration of IL-3 plus G-CSF shows a generalized myeloid hyperplasia with a synergistic increase in mature neutrophils as compared with IL-3 or G- CSF alone. Daily injection of IL-3 plus G-CSF induced a significant decrease in erythroid, lymphoid, and eosinophilic marrow precursors, possibly owing to a myelophthisic effect of the myeloid hyperplasia and despite the fact that IL-3 alone induced a significant erythroid hyperplasia.

The similar proximal and distal attachments to the stereom of primary spine ligament in the echinoid Eucidaris tribuloides are described, from thin sections and SEM studies on frozen and fractured spine articulations and ligaments from decalcified material. The orthogonal structure of the general stereom is modified on the attachment zones where bundles of collagen cylinders enter approximately hexagonally arranged channels. Straps of collagen extend in parallel series between adjacent bundles via regularly placed ports and collagen loops rather than non-striated 'tendons' pass over skeletal trabeculae. The regular pattern of collagen straps is most evident on the proximal and distal attachment zones. Mechanical features of the non-adhesive mode of attachment are considered, together with similarities and differences between insertion of muscle cells and mutable collagenous tissue (ligament) in echinoderms.

Inherited hearing impairment is a highly heterogeneous group of disorders.1,2 In a majority of cases (about 70%), the hearing loss is non-syndromic, that is, it is not associated with any other clinical feature. It can be transmitted following autosomal (recessive or dominant), X linked, or maternal inheritance patterns. In the nuclear genome, more than 70 loci have been reported to be involved in non-syndromic hearing impairment, and 27 genes have been isolated from their critical intervals.2 In addition, a number of different mutations in several genes of the mitochondrial genome are responsible for hearing impairment.3,4 Some of these mutations result in a variety of additional clinical features in diverse organs. Mitochondrial syndromic hearing loss includes Kearns-Sayre syndrome (MIM 530000), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MIM 540000), MERRF (myoclonus epilepsy and ragged red fibres; MIM 545000), and MIDD (maternally inherited diabetes mellitus and deafness; MIM 520000).3–5 In other cases, intrafamilial and interfamilial phenotypic variation is observed, a mutation causing syndromic or non-syndromic hearing impairment in different patients. This is the case for the 7445A>G mutation, causing hearing loss and palmoplantar keratoderma,6–10 and for 7472insC, responsible for hearing loss and neurological disorders (myoclonus epilepsy, ataxia, and cognitive impairment).11–14 Finally, other mutations have been associated so far only with hearing loss. These include mutations 7510T>C15 and 7511T>C16 in the tRNASer(UCN) gene, and 1095T>C17 and 1555A>G18 in the gene for the 12S rRNA. This last mutation is responsible for a dual phenotype, since it also confers increased susceptibility to the ototoxic action of aminoglycoside antibiotics.18 Most of the mutations causing maternally inherited non-syndromic …

Background Continuous Renal Replacement Therapies (CRRT) are the treatment of choice for critically ill children with Acute Renal Injury. Hypotension after starting CRRT is frequent but there are no studies that have analysed their incidence and importance. Patients and methods A prospective, observational study was performed including critically ill children treated with CRRT between October 2009 and December 2013. Hemodynamic data and connection characteristics were collected before, during and 60 min after CRRT circuit connection. Hypotension with the connection was defined as a decrease in mean arterial pressure >20% from baseline and/or intravenous fluid expansion and/or if increase in vasopressors was required. Results 161 connections in 36 children (median age 18.8 months) were analysed. 28 patients (77.8%) were in the postoperative period of cardiac surgery, 94% on mechanical ventilation and 86.1% with vasopressors. The circuit prime was discarded in 8.7% of connections, the heparinised prime was infused in 18% and the circuit was previously primed witha colloid (albumin in 77.5%) or crystalloid without heparine in 73.3%. Hypotension occurred in 49.7% of connections with a median of 5 min after the beginning. In 38.5% of the connections fluid expansion was required and in 12.4% vasopressors were increased. There was no hypotension relation to age or weight. Previous priming of the circuit reduced the frequency of hypotension to 44.6% vs. 71.4% (p = 0.004). Conclusions Hypotension after CRRT connection is very frequent in critically ill children. Priming the circuit improves hemodynamic tolerance of the connection.

Background The Synovitis, Acne, Pustulosis, Hyperostosis and Osteitis (SAPHO) syndrome is a rare autoinflammatory disorder of skin and bone that can be classified with the inflammatory spondyloarthropathies (1). Genetic predisposing factors for SAPHO syndrome have been long suspected because of (i) reported familial clustering of the syndrome - although most cases are sporadic - (2), and (ii) the existence of two mouse models of the syndrome (the cmo and Lupo strains), each with a different spontaneous recessive mutation in the Pstpip2 gene (3,4). Previous studies excluded a role for the genes PSTPIP2 , LPIN, NOD2 , IL1RL2 , IL1RN , IL36RN and SIGLEC15 in the pathogenesis of the syndrome (5,6). Objectives Identify and validate candidate genes involved in the pathogenesis of familial SAPHO syndrome by performing whole-exome sequencing (WES) of affected subjects and unaffected relatives. Methods We investigated a cohort of 44 SAPHO subjects that includes 5 families segregating the syndrome. After obtaining written informed consent, we extracted DNA from peripheral blood samples by using automated magnetic-bead technology (Chemagen). Exomes captured with Agilent SureSelect V5+UTR kit were subjected to paired-end sequencing on an Illumina HiSeq200 sequencer (Sistemas Genόmicos). Candidate genes and putatively pathogenic variants were identified by using Variant Analysis software (Ingenuity). Validation was undertaken by Sanger sequencing, segregation analysis and functional assays when appropriate. Results We identified several potentially pathogenic variants in our cohort in genes whose products are potentially involved in the pathogenesis of SAPHO syndrome. In affected subjects we also found variants known to predispose to autoinflammatory/autoimmune disease in genes such as NOD2 / CARD15 . Conclusions WES is a very powerful tool to investigate the genetic factors underlying rare inherited autoinflammatory disorders that cannot be analyzed by classical genetic methods such as positional cloning. References 1. Clin Exp Rheumatol 1988; 6: 109-112. 2. Joint Bone Spine 2007; 74: 123-126. 3. Bone 2006; 38: 41-47. 4. Blood 2006, 107; 3350-3358. 5. J Rheumatol 2010; 37; 401-409. 6. Ann Rheumat Dis 2013; 72 - Suppl 3; 169. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4251

Background We previously reported that ARANESP™ alleviates ACD in a rodent model of peptidoglycan-polysaccharide polymer (PG-APS) mediated inflammation. We report here the further characterisation of this model and the effects of ARANESP™ treatment on ACD. Objectives Methods Immunisation of Lewis rats with PG-APS induces chronic systemic inflammation characterised by relapsing arthritis and hepatic granulomas. Associated with the inflammation is acute, severe anaemia followed by chronic, moderately severe anaemia. As previously established, a 30 μg/kg dose of ARANESP™ every 2 weeks starting day 36 normalised peripheral blood (PB) haemoglobin levels by day 64. Results Acutely anaemic rats had greatly enhanced mean PB reticulocyte counts and greatly reduced mean RBC counts. Mean PB reticulocyte and RBC counts normalised during chronic anaemia, but RBC remained hypochromic and microcytic. Individual anaemic rats had transient increases in serum erythropoietin (EPO) concentrations. However, there was no significant difference in mean EPO concentrations compared to controls, which suggests EPO production was blunted. Histology of day 36 and day 112 anaemic rat spleen sections revealed greatly enhanced iron retention by splenic macrophages. In contrast, bone marrow (BM) macrophages were nearly devoid of iron. Serum iron concentratioans were significantly reduced by day 7 and remained low throughout the study. Interestingly, ARANESP™ treated rats showed decreased iron retention in macrophages and increased serum iron starting day 98. To identify which cytokines may contribute to the chronic anaemia of this model, peritoneal exudate cells (PEC) were isolated and challenged with PG-APS in vitro. Unstimulated PEC produced little or no cytokines. PG-APS challenged PEC from anaemic rats produced IL-1α, TNF-α, and IFN-γ. PG-APS challenged PEC from ARANESP™ treated anaemic rats trended towards reduced levels of these cytokines relative to anaemic PEC cultures. Conclusion In summary, we have shown this ACD model is similar to human ACD. Importantly, ARANESP™ treatment alleviates ACD in this model, thus indicating its potential therapeutic utility for human ACD.

Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.

Objective: To study the evolution of children requiring continuous renal replacement therapy (CRRT) after cardiac surgery and to analyse factors associated with mortality. Material and methods: Prospective observational study between 1996 and 2009 in children after cardiac surgery requiring CRRT. Univariate and multivariate analysis were performed to analyse the influence of each factor on mortality. Results: Of the 1650 post-operatory cardiac surgery patients admitted in the ICU between 1996 and 2009, 81(4,9%) required CRRT. 26% weighed less than 5 kg. Patients with CRRT after cardiac surgery had a mean arterial pressure, creatinine and urea at the beginning of the CRRT, significantly lower than other critically ill children with CRRT, and they needed mechanical ventilation more frequently. There were no significant differences in the mortality risk scores PRISM, PIM y PELOD. Mortality of patients with CRRT after cardiac surgery was higher 43% than the mortality of the rest of patients 29% (p=0,051). Factors associated with mortality were age less than 12 months, weight less than 10 kg, hypotension, high PRIMS score and low creatinine and urea at the beginning of the CRRT, and hemofiltration (without dialysis). In the multivariate study the only factor associated with mortality was the hypotension at the beginning of CRRT (Hazard Ratio: 4.01; CI 95%:1.2-13.4; p=0.024). Conclusions: A 5 % of children after cardiac surgery require CRRT. Mortality of these patients is higher than other critically ill children who require CRRT. Hypotension at the beginning of the CRRT is the most important factor related to mortality.

Auteur(s) : Olivier Join-Lambert1,2, Sylvie Fraitag3, Florence Ribadeau-Dumas1,4, Anne-Sophie Leguern1, Sylvie Behillil1, Francisco-J Del Castillo1,5, Paul-Henri Consigny1, Frederic Auquier6, Francois Eb6, Henri Sevestre6, Olivier Lortholary1,4, Xavier Nassif1,2, Aude Nassif1 1Centre d’Infectiologie, Necker-Pasteur, Institut Pasteur, Centre Medical, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France 2Laboratoire de Microbiologie, Hopital Necker-Enfants malades, 149 rue de [...]

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.