Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Cell proliferation and apoptosis during histogenesis of the guinea pig and rabbit cerebellar cortex

Cell proliferation and apoptosis are essential for development of the nervous system. In this study we have investigated the histogenesis of the cerebellar cortex in guinea pig (a precocial species) and rabbit (an altricial species) at different stages of pregnancy and postnatal life. Proliferating cells were identified after labeling with antibodies against the proliferating cell nuclear antigen (PCNA) and/or the Ki-67 antigen. Apoptotic cells were visualized in situ by the TUNEL method and by immunodetection of cleaved caspase 3 and 9. In guinea pigs, both proliferating and apoptotic cells were detected during pre-natal life (E0-E40). Conversely, cell proliferation and apoptosis in rabbits were temporally restricted to early postnatal weeks (P0-P20). In both species cell proliferation was mainly linked to differentiation and migration of the granule cells. In both species, the majority of cells undergoing programmed cell death likely corresponded to granule cells. They were mainly detected in the external granular layer, and were by far more common than previously reported in other locations of the postnatal brain. This study shows that apoptosis is a shared process of cell death during cerebellar development in both altricial and precocial animals, and that there is a direct spatial and temporal correlation between cell proliferation and death in two mammals with different time tables in cerebellar maturation.     

Facial hemangioma and malformation of the cortical development: a broadening of the PHACE spectrum or a new entity?

Facial hemangioma is usually isolated but its association with craniocervical arterial anomalies and structural brain malformations is well known. The acronym PHACE syndrome (posterior fossa malformation, facial hemangiomas, arterial anomalies, cardiac/aortic anomalies, and eye abnormalities) has been used to indicate that disorder in which brain anomalies are mainly represented by the Dandy-Walker malformation. We report on a 10-month-old boy affected by facial hemangioma and a complex cortical dysplasia located in the left frontal region. The lesion was characterized by a deeply infolding pachygyric cortex and a band of gray matter lining the wall of the lateral ventricle. The entire left cerebral hemisphere appeared hypoplastic. No anomalies of the posterior fossa structures or cardiac/aortic malformations were present. An overlapping clinical/pathological pattern was previously reported in another patient with facial hemangioma and cerebrovascular anomalies. These observations seem to indicate that the facial hemangiomas may be associated with disorders of the cortical development.     

Interleukin-7-engineered mesenchymal cells: in vitro effects on naive T-cell population.

T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.     

Sodium channel heterologous expression in mammalian cells and the role of the endogenous beta1-subunits

Sodium currents in cell lines transfected with the sole alpha-subunit, or constitutively expressing sodium channels, have an inactivation that is always prevalently mono-exponential. Differently, expression of alpha-subunit in Xenopus oocytes exerts slow inactivating currents with biphasic decay, while simultaneous co-transfection of alpha and beta1 restores a mono-exponential (normal) inactivation. A hypothesis for such differences is that an endogenous presence of beta1 or beta1-alternative splicing, beta1A, in cells could account for the normal inactivation. To test this hypothesis and to evaluate the role for the beta1A, we inhibited the expression of beta1/beta1A by antisense oligonucleotides on Nav1.4-transfected human embryonic cell line 293 (HEK) cells. Reduction of beta1/beta1A produces no significant functional effects in Nav1.4-HEK. This result invalidates the hypothesis that the lack of slow-mode in cell lines is simply due to a constitutive expression of beta1/beta1A.     

Coexpression of Aspartic Proteinases and Human Leukocyte Antigen-DR in Human Transplanted Lung

Aspartic proteinases have recently been shown to be implicated in antigen processing. We explored the expression of two aspartic proteinases, cathepsins E and D, and of human leukocyte antigen-DR (HLA-DR) molecules in a consecutive series of 80 transbronchial biopsies from transplanted lungs. For controls, we studied five normal donor lungs (not suitable for transplantation on account of thoracic trauma) and macroscopically normal areas of three cancer-affected lungs. Two of the five unsuitable donor lungs showed minimal inflammatory changes. Macroscopically normal samples from the three cancerous lungs showed mild and focal inflammatory infiltrates. In histologically normal lungs, HLA-DR expression was limited to professional antigenpresenting cells. Macroscopically normal lung samples with minimal inflammatory changes from both donor and cancer lungs showed variable HLA-DR expression by alveolar and bronchial epithelial cells and by endothelial cells. All transplanted lung biopsies showed HLA-DR expression by epithelial (alveolar and bronchial) and endothelial cells, with a trend for increased positivity in acute rejection. Cathepsin E was restricted to Clara and to rare bronchus-associated lymphoid tissue-related epithelial cells in histologically normal lung samples, whereas minimal de novo cathepsin E expression by rare alveolar pneumocytes was noted in control lung samples exhibiting minimal inflammatory changes. In all transplanted lung biopsies, cathepsin E was diffusely expressed de novo by hyperplastic alveolar epithelial cells, regardless of the presence or degree of rejection. Cathepsin D was expressed only by alveolar macrophages and by ciliated bronchial cells of normal, minimally inflamed, and transplanted lungs. In transplanted lung, Clara cells and several hyperplastic alveolar pneumocytes coexpressed HLA-DR and cathepsin E, whereas all alveolar macrophages and a few ciliated cells coexpressed cathepsin D and HLA-DR The present investigation suggests that the de novo expression of cathepsin E and HLA-DR by hyperplastic alveolar pneumocytes of transplanted lung may be crucial for antigen processing and presentation to recipient competent T cells, and thus for the triggering of the immune-inflammatory cascade that leads to rejection.