Clinical, immunological and molecular characteristics of 37 Iranian patients with X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency characterized by an early onset of recurrent bacterial infections, a profound deficiency of all immunoglobulin isotypes and a markedly reduced number of peripheral B lymphocytes. Eighty-five percent of the patients with this phenotype have mutations in Bruton's tyrosine kinase (BTK) gene. METHODS: To provide an informative outlook of clinical and immunological manifestations of XLA in Iran, 37 Iranian male patients with an age range of 1-34 years, followed over a period of 25 years, were studied. Twenty-four of the 37 patients were screened for BTK gene mutation using PCR-SSCP followed by direct sequencing. BTK protein expression assay was done by flow cytometry in 9 families. RESULTS: All patients first presented with infectious diseases, the most common of which were respiratory tract infections. Eighteen different mutations were identified, 13 of which were novel: IVS1+5G>C, 1896G>A, 349delA, 1618C>T, 1783T>C, 2084A>G, 1346delT, 1351delGAG, 587A>G, IVS14-1G>A, IVS3+2T>C, 1482G>A, 1975C>A. CONCLUSION: The fact that we found a great number of novel mutations in a relatively limited number of patients underlines the heterogeneity of BTK mutations in the Iranian population. The large number of new mutations indicates that extended studies in this region would be rewarding.

Immature neuronal phenotype derived from mouse skin precursor cells differentiated in vitro.

Recent findings indicate that skin-derived precursor cells (SKPs) of mouse dermis can differentiate in cells with neuronal-like morphology. However, direct evidence supporting the establishment of functional phenotype is missing. In the present study, SKP cells were obtained using published in vitro techniques and studied at 14- to 21-day differentiation, when neuronal-like morphology was observed. The experiment was repeated 39 times. Co-cultures with cortical astrocytes were also used to enhance the process of neural differentiation. Expression of GAP43 and light-chain MAP-2(c) but not markers of dendritic and synaptic terminal differentiation such as heavy-chain MAP-2(ab) and synapsin were observed. Voltage-clamp electrophysiology recordings showed potassium currents, but neither action potentials generation nor electrotonic response to exogenous administration of nicotine or kainic acid. These observations suggest that mouse SKPs do not differentiate into mature functional neurons, at least using the published methodologies for in vitro differentiation.

Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line.

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.

Opposing roles for NF-kappa B/Rel factors p65 and c-Rel in the modulation of neuron survival elicited by glutamate and interleukin-1beta.

The nuclear transcription factors NF-kappaB/Rel have been shown to function as key regulators of either cell death or survival in neuronal cells. Here, we investigated whether selective activation of diverse NF-kappaB/Rel family members might lead to distinct effects on neuron viability. In both cultured rat cerebellar granule cells and mouse hippocampal slices, we examined NF-kappaB/Rel activation induced by two opposing modulators of cell viability: 1) interleukin-1beta (IL-1beta), which promotes neuron survival and 2) glutamate, which can elicit toxicity. IL-1beta produced a prolonged stimulation of NF-kappaB/Rel factors by inducing both IkappaBalpha and IkappaBbeta degradation. Glutamate produced a delayed and transient activation of NF-kappaB/Rel, which was associated with a brief loss of IkappaBalpha. Moreover, IL-1beta activated the p50, p65, and c-Rel subunits of NF-kappaB/Rel, whereas glutamate activated only the p50 and p65 proteins. The inhibition of NF-kappaB/Rel protein expression by antisense oligonucleotides in cerebellar granule cells showed that p65 was involved in glutamate-mediated cell death, whereas c-Rel was essential for IL-1beta-preserved cell survival. Furthermore, the depletion of c-Rel in cultured neurons as well as in the hippocampus from the c-Rel(-/-) mouse converted the IL-1beta effect into toxicity. These findings suggest that, within a single neuron, the balance between cell death and survival in response to external stimuli may rely on the activation of distinct NF-kappaB/Rel proteins.