It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.
The exceptional diploid number (2n=48) of the proboscis monkey (Nasalis larvatus) has played a pivotal role in phylogenies that view the proboscis monkey as the most primitive colobine, and a long-isolated genus of the group. In this report we used molecular cytogenetic methods to map the chromosomal homology of the proboscis monkey in order to test these hypotheses. Our results reveal that the N. larvatus karyotype is derived and is not primitive in respect to other colobines (2n=44) and most other Old World monkeys. The diploid number of 2n=48 can be best explained by derived fissions of a segment of human chromosomes 14 and 6. The fragmentation and association of human chromosomes 1 and 19 as seen in other Asian colobines, but not in African colobines, is best explained as a derived reciprocal translocation linking all Asian colobines. The alternating hybridization pattern between four segments homologous to human chromosomes 1 and 19 on N. larvatus chromosome 6 is the result of the reciprocal translocation followed by a pericentric inversion. N. larvatus shares this pericentric inversion with Trachypithecus, but not with Pygathrix. This inversion apparently links Nasalis and Trachypithecus after the divergence of Pygathrix. The karyological data support the view that Asian colobines, including N. larvatus, are monophyletic. They share many linking karyological features separating them from the African colobines. The hybridization pattern also suggests that Nasalis is nested within Asian Colobines and shares a period of common descent with other Asian colobines after the divergence of Pygathrix.
Cases of tetrasomy 12p and trisomy 12p are known to be associated with specific phenotypic abnormalities well described in the literature. Here, we report on the unusual case of a partial tetrasomy 12p found in an affected patient and in a mosaic constellation in the patient's mother, who showed no phenotypic abnormality. The index patient was a 16-year-old boy with clinical features similar to the "trisomy 12p syndrome" including mental retardation, macrocephaly, a short nose with anteverted nostrils, and a broad protruding lower lip. G-banding analysis and fluorescence in situ hybridization (FISH) experiments using locus specific YAC DNA probes revealed a derivative chromosome 12 with a partial triplication of the short arm with an inverted copy, flanked by two direct copies. Chromosome analyses in parental lymphocytes showed a chromosomal mosaicism in the phenotypically normal mother, with 12% cells exhibiting the same partial tetrasomy 12p as detected in her son. The allelic pattern of short tandem repeats (STR) in the mother's blood DNA showed that a chimerism can be excluded with high probability. To our knowledge, this is the first report of intrachromosomal triplication on chromosome 12, as well as partial tetrasomy 12p mosaicism. Moreover, as a consequence of the chromosomal aberration in the son it can be concluded that a gonadal mosaicism is present in the mother.
The human male specific expressed gene families CDY and DAZ are known to be repetitively clustered in the Y-specific region of the human Y chromosome. Comparative FISH-mapping of DNA clones specific for CDY and DAZ resulted in a Y-specific but diverse signal pattern within the non-recombining region of the Y-chromosomes of human and great apes. It can be concluded that the non-recombining part of the Y-chromosomes including CDY and DAZ, was exposed to species-specific amplifications, diversifications and rearrangements. Evolutionary fast fixation of any of these variations was possible as long as they did not interfere with male fertility.
A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)n. The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.
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