Exclusion of Treacher Collins Franceschetti syndrome in a subject with tetralogy of Fallot and cryptorchidism

Genotyping with flanking DNA markers was used to ascertain Treacher Collins Franceschetti syndrome (TCOF1) in a subject affected by tetralogy of Fallot and cryptorchidism. The proband's family consisted of a father and sister who were affected by the disease, and a healthy mother. Since cardiac malformation and cryptorchidism have been associated with the TCOF1 syndrome, the proband was suspected to be a carrier of the mutated gene. Microsatellite markers D5S527, SPARC and D5S519, which previously mapped the TCOF1 gene within a 2.1-cM interval on chromosome 5 (5q32–33.1), were used to follow the transmission of the TCOf 1 mutated locus. Flanking markers D5S519 and D5S527 were informative and enabled us to exclude inheritance of a TCOF1 mutation to the proband, while showing that cardiac malformation and cryptorchidism were unrelated in mis patient.     

Altered blood-brain barrier development in dystrophic MDX mice

In order to ascertain whether the alterations of the blood-brain barrier (BBB) seen in adult dystrophic mdx-mice [Glia 42 (2003) 235], a human model of Duchenne muscular dystrophy (DMD), are developmentally established and correlated with other dystrophin isoforms which are localized at the glial-vascular interface, we used immunocytochemistry to investigate the expression of dystrophin isoforms (Dp71) during BBB development in mdx fetuses and in adult mice. Parallelly, we used Western blot, immunocytochemistry and immunogold electron microscopy to analyze the expression of the zonula occludens (ZO-1), aquaporin-4 (AQP4) and glial fibrillary acidic (GFAP) proteins as endothelial and glial markers, and we evaluated the integrity of the mdx BBB by means of intravascular injection of horseradish peroxidase (HRP). The results show reduced dystrophin isoforms (Dp71) in the mdx mouse compared with the control, starting from early embryonic life. Endothelial ZO-1 expression was reduced, and the tight junctions were altered and unlabeled. AQP4 and GFAP glial proteins in mdx mice also showed modifications in developmental expression, the glial vascular processes being only lightly AQP4- and GFAP-labeled compared with the controls. Confocal microscopy and HRP assays confirmed the alteration in vessel glial investment, GFAP perivascular endfoot reactivity being strongly reduced and BBB permeability increasing. These results demonstrate that a reduction in dystrophin isoforms (Dp71) at glial endfeet leads to an altered development of the BBB, whose no-closure might contribute to the neurological dysfunctions associated with DMD.     

Two new mutations and three novel polymorphisms in the<Emphasis Type="Italic"> RB1</Emphasis> gene in Ecuadorian patients

RB1 is the gene responsible for retinoblastoma, the most common malignant intraocular tumor of infancy and early childhood. There are no reports about this gene in Ecuadorian populations, and only a few studies have been published in Latin America about this subject. There is a spectrum of more than 370 mutations described in the RB1 gene mutation database (http://www.d-lohmann.de/Rb/mutations.html), and alterations have been found in 25 of the 27 exons. During the exon-by-exon analysis of 31 tumor and blood samples from Ecuadorian patients, we found two new mutations and three novel polymorphisms. One of the polymorphisms is located in intron 26 where no alterations of the gene have been described previously. The polymorphisms were found in all of the patients tumor samples, but not in normal population, suggesting there might be a relationship between these polymorphisms and the development of retinoblastoma in the Ecuadorian population.The nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY243567, AY260472, AY260473, AY273783     

Human cytomegalovirus replication correlates with differentiation in a hematopoietic progenitor cell line and can be modulated by HIV-1

Summary Human cytomegalovirus (HCMV) infection of a CD34+ hematopoietic progenitor cell line (TF1) was studied before and after TPA differentiation. TF1 cells were found to be infected but the virus does not replicate, while differentiated TF1 cells can be infected and allow HCMV complete replication. In the same system we studied the interaction between HCMV and HIV and found that while contact between HIV gp 120 and the HCMV-infected cell has an inhibitory effect, exogenous Tat protein stimulates HCMV replication. The interaction between HCMV and HIV in hematopoietic progenitor cells is complex and depends on several factors that can have opposite effects.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO)at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5)Chinese hamster ovary cells were determined to investigate theeffect of DNA repair on the nature of induced mutations. TheUV5 cell line is three times more sensitive to 4NQO than theAA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct,which is considered to be the most toxic and mutagenic adductin Escherichia coli, is poorly repaired. The molecular natureof 30hprt mutants isolated from AA8 and 20 isolated from UV5cells was determined by sequence analysis of in vitro amplifiedhprtcDNA. Both similarities and differences emerged. In bothcell lines we found that (i) 4NQO is basically a base substitutionmutagen acting almost exclusively at G residues and (ii) G transversionsare prevalent over G transitions in both cell lines, independentlyfrom the ability to repair dGuo-N2-AQO. A high proportion (13/25)of splice mutations was observed in AA8 cells, statisticallydifferent (P < 0.04, Fisher‘s exact test) from theincidence of splice mutants in UV5 cells (4/20). In AA8 mutants,all but two of the point mutations were due to lesions localizedon the non-transcribed strand, suggesting preferential repairof the transcribed strand. Compared with AA8, the proportionof mutants due to lesions present on the transcribed strandwas higher in UV5 cells, as expected if a preferential repairmechanism was impaired in the sensitive cell line. Our dataare consistent with the molecular defect in DNA repair recentlycharacterized in UV5. ³To whom correspondence should be addressed     

Vestibular and audiological findings in the Alport syndrome

Alport syndrome (AS) is caused by mutations in collagen IV, which is widespread in the basement membranes of many organs, including the kidneys, eyes, and ears. Whereas the effects of collagen IV changes in the cochlea are well known, no changes have been described in the posterior labyrinth. The aim of this study was to investigate both the auditory and the vestibular function of a group of individuals with AS. Seventeen patients, aged 9–52, underwent audiological tests including pure‐tone and speech audiometry, immittance test and otoacoustic emissions and vestibular tests including video head impulse test, rotatory test, and vestibular evoked myogenic potentials. Hearing loss affected 25% of the males and 27.3% of the females with X‐linked AS. It was sensorineural with a cochlear localization and a variable severity. 50% of the males and 45.4% of the females had a hearing impairment in the high‐frequency range. Otoacoustic emissions were absent in about one‐third of the individuals. A peripheral vestibular dysfunction was present in 75% of the males and 45.4% of the females, with no complaints of vertigo or dizziness. The vestibular impairment was compensated and the vestibulo‐ocular reflex asymmetry was more evident in rotatory tests carried out at lower than higher speeds; a vestibular hypofunction was present in all hearing impaired ears although it was also found in subjects with normal hearing. A posterior labyrinth injury should be hypothesized in AS even when the patient does not manifest hearing disorders or evident signs of renal failure.     

Tuning of innate immunity and polarized responses by decoy receptors

After the identification of the interleukin (IL)-1 type II receptor as the prototype, decoy receptors have been identified for a number of members of the IL-1/IL-18, TNF, IL-10 and IL-13 receptor families. Moreover, the silent receptor D6 is a promiscuous decoy and scavenger receptor of inflammatory chemokines. The IL-1 decoy receptor is regulated by pro- and anti-inflammatory signals and its levels may serve as a readout of the activation of anti-inflammatory pathways, for instance by glucocorticoid hormones. Decoy receptors represent a strategy to tune inflammatory and polarized adaptive responses.     

Diltiazem impairs maturation and functions of human dendritic cells

The aim of this study was to define the effects of diltiazem, a calcium antagonist drug used in cardiology and in clinical transplantation, on the differentiation and maturation of human dendritic cells (DC). Herein, we demonstrate that diltiazem, in association with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), induces monocytes to differentiate into cells with many of the characteristic of DC. However, diltiazem-induced DC express high levels of mannose receptor and Fc gamma RII and, consequently, manifest a higher endocytic activity compared with GM-CSF+IL-4-induced DC. Importantly, diltiazem-induced DCs have an impaired responsiveness to lipopolysaccharide and CD40 ligand because they produce decreased levels of IL-12 and reveal a reduced ability to stimulate alloreactive T-cell responses as well as in inducing interferon-gamma producing Th1 cells. These effects may contribute to a decreased DC-dependent T-cell activation and may help to explain the immunoregulatory function of diltiazem and its effectiveness in preventing transplant rejection.     

Immunomagnetic cell selection performed for HLA haploidentical transplants with the CliniMACS device: effect of additional platelet removal on CD34+ cell recovery

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein-Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.