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PubMed1996

Primed in situ labeling (PRINS). A fast method for in situ labeling of nucleic acids.

PRimed IN Situ labeling (PRINS) is a fast and sensitive alternative to fluorescence in situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.

Chromosomes, Human↗
PubMed1986

An anesthesia information system for monitoring and record keeping during surgical anesthesia.

We have developed an anesthesia information system (AIS) that supports the anesthesiologist in monitoring and recording during a surgical operation. In development of the system, emphasis was placed on providing an anesthesiologist-computer interface that can be adapted to typical situations during anesthesia and to individual user behavior. One main feature of this interface is the integration of the input and output of information. The only device for interaction between the anesthesiologist and the AIS is a touch-sensitive, high-resolution color display screen. The anesthesiologist enters information by touching virtual function keys displayed on the screen. A data window displays all data generated over time, such as automatically recorded vital signs, including blood pressure, heart rate, and rectal and esophageal temperatures, and manually entered variables, such as administered drugs, and ventilator settings. The information gathered by the AIS is presented on the cathode ray tube in several pages. A main distributor page gives an overall view of the content of every work page. A one-page record of the anesthesia is automatically plotted on a multicolor digital plotter during the operation. An example of the use of the AIS is presented from a field test of the system during which it was evaluated in the operating room without interfering with the ongoing operation. Medical staff who used the AIS imitated the anesthesiologist's recording and information search behavior but did not have responsibility for the conduct of the anesthetic.

Anesthesia Department, Hospital↗
PubMed1997

Detection of Numerical Alterations of Chromosomes 3, 7, 17 and X in Low-grade Intracystic Papillary Tumors of the Breast by Multi-color Fluorescence In Situ Hybridization.

Intracystic papillary tumors of the breast comprise intraductal papillomas and low-grade papillary carcinomas. To determine chromosomal alterations that may be characteristic of these tumors, chromosomes 3, 7, 17 and X were examined in 10 intraductal papillomas and 14 low-grade papillary carcinomas using multicolor fluorescent in situ hybridization. In papillry carcinomas, numerical/structural alterations in chromosome 17 and the accumulation of numerical alterations in chromosomes 3, 7 and X were detected in 21%(3/14) and 15%(2/13) of the cases, respectively. These changes were not detected in papillomas. Loss of chromosome 17p was detected in only one of the 11 DNA-diploid carcinomas, whereas alterations in all four chromosomes were present in a case of DNA-aneuploid carcinoma. Numerical alterations of chromosomes 3, 7, 17 and X do not appear to be involved in the development of low-grade papillary carcinomas with DNA diploidy, but rather accumulate during the formation of DNA-aneuploid carcinoma cells in association with other chromosome alterations.

Journal Article↗
PubMed1999

[Tattoo removal Q-switched ruby laser (694 nm) and the Q-switched Nd:YAG laser (532 and 1064 nm). A retrospective study].

In a retrospective study 47 patients with 68 amateur and 25 professional tattoos were examined, considering clearance of tattoo pigments and the frequency of side effects after finished treatments with the Q-switched ruby (694 nm) and the Q-switched Nd:YAG laser (532 and 1064 nm). Black amateur tattoos were found to lighten faster than professional tattoos (13.2 and 18.6 treatments respectively). For the clearance of multicolored tattoos (amateur or professional), similar numbers of laser treatments were needed. Green pigments were removed with the Q-switched ruby laser and red pigments with the frequency-doubled Nd:YAG laser (532 nm). Compared to the Q-switched Nd:YAG laser (1064 nm), the Q-switched ruby laser and the frequency-doubled Nd:YAG laser (532 nm) more often caused blistering and transient hypopigmentation (5.4% and 7.5% vs. 1.1% (blisters); 8.6% and 15.1% vs. 4.3% (hypopigmentation).

Adult↗
PubMed2006

Nano-oncology: drug delivery, imaging, and sensing.

Innovation in the last decade has endowed nanotechnology with an assortment of tools for delivery, imaging, and sensing in cancer research-stealthy nanoparticle vectors circulating in vivo, assembled with exquisite molecular control, capable of selective tumor targeting and potent delivery of therapeutics; intense and photostable quantum dot-based tumor imaging, enabling multicolor detection of cell receptors with a single optical excitation source; arrays of semiconducting nanowire and carbon nanotube sensor elements for selective multiplexed sensing of cancer markers without the need for probe labeling. These rapidly emerging tools are indicative of a burgeoning field ready to expand into medical applications. This review attempts to outline most of the current nanoparticle toolset for therapeutic release by liposomes, dendrimers, smart polymers, and virus-based systems. Advantages of nanoparticle-based imaging and targeting by use of nanoshells and quantum dots are also explored. Finally, emerging nanoelectronics-based sensing and a global discussion on the utility of each nanoparticle system addresses their fundamental advantages and shortcomings in cancer research.

Biosensing Techniques↗
PubMed2006

Anthozoa red fluorescent protein in biosensing.

The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.

Animals↗
PubMed2003

Rapid turnover of the CD8(+)CD28(-) T-cell subset of effector cells in the circulation of patients with head and neck cancer.

CD8(+) T cells in the circulation of patients with head and neck cancer (HNC) were previously shown to be significantly more sensitive to, and preferentially targeted for, apoptosis than CD4(+) T cells (Hoffmann et al., Clin Cancer Res, 8:2553-2562, 2002). To distinguish global from CD8(+) subset-specific apoptosis, we studied Annexin-binding to naïve, memory, and effector subsets of CD8(+) cells by multicolor flow cytometry. Age-related changes in naïve and effector CD8(+) cell subsets were observed in patients and normal controls (NC). The frequencies of naïve (CD28(+)CD45RO(-)) CD8(+) T cells were lower and those of memory (CD28(+)CD45RO(+)) and effector (CD28(-)) CD8(+) T cells significantly higher in the circulation of HNC patients relative to age-matched NC. Among CD8(+) T cells, the CD28(-) effector cell subset contained the highest proportion of Annexin-binding cells, while the naïve CD28(+)CD45RO(-) subset contained the lowest. This suggested a high turnover rate of the CD8(+)CD28(-) effector cell subset in patients with HNC, which was being compensated by a rapid transition of naïve CD8(+) T cells to the effector cell pool. Following tumor resection, the frequency of CD8(+)CD28(-) T cells normalized in the patients, an indication that the presence of tumor had an influence on the size of CD8(+)CD28(-) T-cell pool. Ex vivo, in mixed lymphocyte-tumor cultures (MLTC) with semiallogeneic T cells as responders, CD8(+)CD28(-) T cells could be generated from CD8(+)CD28(+) cells by repeated stimulations with tumor cells. These CD8(+)CD28(-) effector cells lysed the tumor, produced IFN-gamma in response to the tumor, and strongly expressed granzyme B. Thus, the high rate of their apoptosis in the circulation of patients with HNC might be expected to contribute to tumor progression. However, the ex vivo generation of this cell subset was suppressed by strong CD28/B7 ligation or by overexpresson of MHC molecules on tumor cells, suggesting that adequate costimulation is necessary for protection from apoptosis. It appears that interactions of immune and tumor cells might determine the fate of this terminally differentiated effector cell subset.

Adult↗
PubMed2005

Maturation of circulating dendritic cells and imbalance of T-cell subsets in patients with squamous cell carcinoma of the head and neck.

Interactions between dendritic cells (DCs) and T cells play a pivotal role in the regulation and maintenance of immune responses. In cancer patients, various immunological abnormalities have been observed in these immune cells. Here, we investigated proportions and the phenotype of DCs and the cytokine profile of T-cell subsets in the peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), using multicolor flow cytometry. The percentage of myeloid (CD11c(+)), but not plasmacytoid (CD123(+)) DCs, was significantly lower (P<0.05) and expression of HLA-DR was significantly decreased in total and myeloid DCs of cancer patients compared to healthy donors. Simultaneous analyses of T-cell subsets in the patients' circulation showed significantly increased proportions of CD4(+) T cells expressing Th1 and Th2 cytokines after ex vivo stimulation without any skewing in the Th1/Th2 ratio. The relative level of HLA-DR expression on myeloid or total DCs positively correlated with the Th1/Th2 ratio (P<0.01), and the proportion of total circulating DCs was inversely correlated with that of regulatory CD4(+)CD25+() T cells (P<0.01). These results suggest that the decreased proportion of circulating DCs and decreased HLA-DR expression in DCs may have a major impact on systemic immune responses in patients with SCCHN.

Adult↗
PubMed1995

[Inflammatory pseudotumor of the liver. Morphologic and cytophotometry studies and differential diagnosis].

Inflammatory pseudotumors of the liver are extremely rare. Worldwide only 50 cases have been described so far. In lung tissue, their incidence is higher, with 119 cases being published. Etiological factors are still a matter of debate; reactive inflammatory processes have recently been proved by immunohistological and cytophotometric studies. This seems to be true for both liver and lung locations of inflammatory pseudotumors. Considerable morphological heterogeneities, conspicuously varying from one case to another, reflect the numerous synonyms that have been created for this condition, i.e., plasma cell granuloma, xanthoma, fibroxanthoma, histiocytoma, plasmacytoma, solitary "mast cell tumor" and pseudoneoplastic pneumonia, just to number a few of them. This multicolored morphology explains the difficulties in histologic diagnosis, especially if needle biopsies or frozen sections are submitted to pathology. Differential diagnosis of an inflammatory pseudotumor of the liver needs to include fibrohistiocytic neoplasia if spindle-cell-shaped areas are included or, on the other hand, parenchymal infiltrates of the nodular sclerosing type of Hodgkin's granuloma. Immunohistological investigations do not allow final decisions since spindle-shaped cells within pseudotumours can express vimentin and/or desmin as well. Such histological cases should always include the excision of a piece of liver tissue of sufficient size to comfortably allow for the recognition of the multicellular composition and morphological heterogeneity of this process.

Adult↗
PubMed2005

Space radiation does not induce a significant increase of intrachromosomal exchanges in astronauts' lymphocytes.

Chromosome aberration analysis in astronauts has been used to provide direct, biologically motivated estimates of equivalent doses and risk associated to cosmic radiation exposure during space flight. However, the past studies concentrated on measurements of dicentrics and translocations, while chromosome intrachanges (inversions) have never been measured in astronauts' samples. Recent data reported in the literature suggest that densely ionizing radiation can induce a large fraction of intrachanges, thus leading to the suspicion that interchanges grossly underestimate the cosmic radiation-induced cytogenetic damage in astronauts. We have analyzed peripheral blood lymphocytes from 11 astronauts involved in short- or long-term space flights in low-Earth orbit using high-resolution multicolor banding to assess the frequency of intrachromosomal exchanges in both pre- and post-flight samples. We did not detect any inversions in chromosome 5 from a total of 2800 cells in astronauts' blood. In addition, no complex type exchanges were found in a total of 3590 astronauts' lymphocytes analyzed by multifluor fluorescence in situ hybridisation. We conclude that, within the statistical power of this study, the analysis of interchanges for biological dosimetry in astronauts does not significantly underestimate the space radiation-induced cytogenetic damage, and complex-type exchanges or intrachanges have limited practical use for biodosimetry at very low doses.

Astronauts↗
PubMed2005

Chromosome reshuffling in birds of prey: the karyotype of the world's largest eagle (Harpy eagle, Harpia harpyja) compared to that of the chicken (Gallus gallus).

Like various other diurnal birds of prey, the world's largest eagle, the Harpy (Harpia harpyja), presents an atypical bird karyotype with 2n=58 chromosomes. There is little knowledge about the dramatic changes in the genomic reorganization of these species compared to other birds. Since recently, the chicken provides a "default map" for various birds including the first genomic DNA sequence of a bird species. Obviously, the gross division of the chicken genome into relatively gene-poor macrochromosomes and predominantly gene-rich microchromosomes has been conserved for more than 150 million years in most bird species. Here, we present classical features of the Harpy eagle karyotype but also chromosomal homologies between H. harpyja and the chicken by chromosome painting and comparison to the chicken genome map. We used two different sets of painting probes: (1) chicken chromosomes were divided into three size categories: (a) macrochromosomes 1-5 and Z, (b) medium-sized chromosomes 6-10, and (c) 19 microchromosomes; (2) combinatorially labeled chicken chromosome paints 1-6 and Z. Both probe sets were visualized on H. harpyja chromosomes by multicolor fluorescence in situ hybridization (FISH). Our data show how the organization into micro- and macrochromosomes has been lost in the Harpy eagle, seemingly without any preference or constraints.

Animals↗
PubMed2000

Structural evolution of the germ line-limited chromosomes in Acricotopus.

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.

Animals↗
PubMed2000

Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains.

The intranuclear arrangement of human chromosome 12 in G0(G1) nuclei from human myeloid leukemia HL60 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using band-specific cosmid clones as probes. Pairs of differently colored cosmids were detected on paraformaldehyde-fixed HL60 nuclei, and their relative positions, internal or peripheral, in individual nuclei were scored. Our results suggest that the intranuclear arrangement of human chromosome 12 is not random. Some chromosomal domains, including the centromere, were located in the periphery of the nucleus, while other domains, including the telomeres, were positioned in the internal areas of the nucleus in GO(G1) cells. Based on the replication banding patterns of metaphase spreads, human chromosome 12 was divided roughly into five large domains. Interestingly, the clones in late replicating domains were preferentially localized in the nuclear periphery, whereas clones in early replicating domains were arranged in the internal areas of the nuclei. The DNA replication timing of each cosmid determined by FISH-based assay did not reflect the replication bands, but an overall profile of the replication timing was relatively correlated with these domains on chromosome 12. These results suggest that the intranuclear arrangement of a human chromosome is correlated with the large-scale replication domains, even before DNA replication.

Cell Nucleus↗
PubMed2006

Micro-array analyses decipher exceptional complex familial chromosomal rearrangement.

Recently there has been an increased interest in large-scale genomic variation and clinically in the consequences of haploinsufficiency of genomic segments or disruption of normal gene function by chromosome rearrangements. Here, we present an extraordinary case in which both mother and daughter presented with unexpected chromosomal rearrangement complexity, which we characterized with array-CGH, array painting and multicolor large insert clone hybridizations. We found the same 12 breakpoints involving four chromosomes in both mother and daughter. In addition, the daughter inherited a microdeletion from her father. We mapped all breakpoints to the resolution level of breakpoint spanning clones. Genes were found within 7 of the 12 breakpoint regions, some of which were disrupted by the chromosome rearrangement. One of the rearrangements disrupted a locus, which has been discussed as a quantitative trait locus for fetal hemoglobin expression in adults. Interestingly, both mother and daughter show persistent fetal hemoglobin levels. We detail the most complicated familial complex chromosomal rearrangement reported to date and thus an extreme example of inheritance of chromosomal rearrangements without error in meiotic segregation.

Child↗
PubMed2005

Evolution versus constitution: differences in chromosomal inversion.

We compared the chromosomal breakpoints of evolutionary conserved and constitutional inversions. Multicolor banding and human-specific bacterial artificial chromosomes were applied to map the breakpoints of constitutional pericentric inversions on human chromosomes 2 and 9. For the first time, we present a high-resolution analysis of the breakpoint regions, which are characterized by gene destitution, co-localization with fragile sites, multitude repeats as well as pseudogenes and, remarkably, a large sequence homology to the opposite breakpoint. In contrast, evolutionary inversion breakpoints lack such extensive cross-hybridizing regions and are often associated with fragile sites of the genome and low-copy repeats. These molecular characteristics gave evidence for different types of inversion formation and indicate that evolutionary inversions cannot originate from constitutional inversions like those of chromosomes 2 and 9. Finally, the constitutional inversion breakpoints were investigated on three different great ape species and on four test persons each bearing the same cytogenetically determined inversion on chromosomes 2 and 9, respectively. Our data indicate the existence of different molecular breakpoints for the two variant chromosomes.

Animals↗
PubMed2000

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies.

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.

Adult↗
PubMed2001

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype.

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.

Animals↗
PubMed2001

AcroM fluorescent in situ hybridization analyses of marker chromosomes.

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.

Adult↗