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Q-switched alexandrite laser treatment (755 nm) of professional and amateur tattoos.

BACKGROUND: Several laser techniques have been proposed for the removal of decorative tattoos. The lasers that have been used most successfully are Q-switched red or near-infrared systems because of their ability to target tattoo pigment selectively with minimal risk of scarring or permanent pigmentary changes. OBJECTIVES: The objectives of this study were to determine the clinical effectiveness of the newest Q-switched system, the alexandrite laser, in removing amateur and professional tattoos and to observe side effects. METHODS: Twenty-four multicolored professional tattoos and 18 blue-black amateur tattoos were treated with the Q-switched alexandrite laser (755 nm, 100 nsec) at 2-month intervals until total clearing was achieved. The 510 nm pulsed dye laser was used to treat tattoos that contained red pigment. RESULTS: Professional tattoos required an average of 8.5 alexandrite laser treatments for total clearance, whereas only 4.6 treatments were necessary to remove amateur tattoos. Red tattoo pigment was successfully treated with an average of two 510 nm pulsed dye laser sessions. No scarring or long-standing pigmentary changes were seen in laser-irradiated skin. CONCLUSION: The Q-switched alexandrite laser is highly effective in removing multicolored professional and amateur tattoos without adverse sequelae. The 510 nm pulsed dye laser was useful in eliminating red tattoo pigment.

Humans↗

Sequential cytogenetic and molecular cytogenetic characterization of an SV40T-immortalized nasopharyngeal cell line transformed by Epstein-Barr virus latent membrane protein-1 gene.

Cytogenetic and molecular cytogenetic analyses were performed on four sublines derived from a newly established, SV40T-immortalized nasopharyngeal (NP) cell line, NP69, with two of the sublines expressing LMP1, an Epstein-Barr virus-encoded gene. A total of seven cytogenetically related subclones were identified, all having highly complex karyotypes with massive numerical and structural rearrangements. Centromeric rearrangements in the form of isochromosomes and whole-arm translocations were prevalent. A cytogenetic sign of gene amplification [i.e., homogeneously staining region (HSR)] was detected at 1q25 in all metaphase cells analyzed. Multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) was used to confirm the karyotypic interpretations. Furthermore, multicolor COBRA-FISH also showed that part of the HSR contained chromosome 20 material. Extensive clonal evolution could be observed by the assessment of karyotypic variation among different subclones and individual metaphase cells. The evaluation of clonal evolution enabled the identification of the temporal order of chromosome aberrations during cell immortalization and malignant transformation. A striking karyotypic similarity was found between sublines expressing LMP1 and an NP carcinoma cell line, with loss of genetic material from chromosome arm 3p being an important recurrent observation. More interestingly, the karyotypic features of NP69 were also similar to those of many epithelial malignancies. Our observations suggest that serial transformation of NP cell lines might provide a useful in vitro model for the study of the multistep neoplastic transformation of NP cells.

Cell Culture Techniques↗

Tetrasomy 12pter-12p13.31 in a girl with partial Pallister-Killian syndrome phenotype.

A dysmorphic patient was shown to carry a small supernumerary marker chromosome. Multicolor, centromere-multicolor and regular FISH experiments proved the marker to be an analphoid 12pter derived isochromosome. Microdissection of the marker followed by reverse painting and array CGH analysis showed that the isochromosome contains approximately 6 Mb of 12pter-12p13.31 derived sequence. This is only the second report of a marker with a neocentromere 12pter and the molecular fine mapping of the duplicated region further refines the 12p region defining the Pallister-Killian syndrome phenotype. In addition, we show the feasibility of using microdissected chromosomes or chromosomal fragments to molecularly map the chromosomal breakpoints on array CGH. This technology may aid in the identification of chromosomal translocation breakpoints.

Abnormalities, Multiple↗

Progenitor analysis of primitive erythropoiesis generated from in vitro culture of embryonic stem cells.

OBJECTIVE: A variety of hematopoietic lineage cells have been produced from embryonic stem (ES) cells, but their differentiation processes have not been elucidated well, especially from the point of view of progenitor analysis. In this study, we utilized our coculture system, in which ES-derived Flk-1+ cells differentiated into TER-119+ primitive erythroid (EryP) cells on OP9 cells, and looked for progenitors in primitive erythropoiesis. MATERIALS AND METHODS: We studied the kinetics of TER-119+ erythroblast generation from Flk-1+ cells by monitoring the expression of TER-119, CD41, VE-cadherin, CD34, and c-kit antigens. Multicolor analysis was performed to detect CD41+TER-119+ cells and the stained cells were sorted to examine their morphology and EryP-producing potential in colony formation. RESULTS: Kinetic studies showed that the CD41+ population appeared early in the coculture and its expression pattern implied a role as an immediate progenitor of TER-119+ EryP cells. Multicolor analysis and colony-formation study supported this notion. Other progenitor markers such as VE-cadherin, CD34, and c-kit did not seem to define an immediate progenitor of EryP cells. One interesting observation is the detection of unique populations, CD41dim and CD41bright, detectable after 48 hours of the coculture. Majority of the CD41dim population progressed to the EryP lineage, whereas the CD41bright population seemingly advanced on a pathway distinct from the CD41dim population. CONCLUSIONS: CD41 expression was a useful marker to trace hematopoietic progenitors in ES-derived differentiation system. In particular, the CD41dim but not CD41bright population could serve as immediate precursors of EryP cells.

Animals↗

CD163 positive subsets of blood dendritic cells: the scavenging macrophage receptors CD163 and CD91 are coexpressed on human dendritic cells and monocytes.

CD163 and CD91 are scavenging receptors with highly increased expression during the differentiation of monocytes into the anti-inflammatory macrophage phenotype. In addition, CD91 is expressed in monocyte-derived dendritic cells (MoDCs), where the receptor is suggested to be important for internalization of CD91-targeted antigens to be presented on the dendritic cell surface for T-cell stimulation. Despite their overlap in functionality, the expression of CD91 and CD163 has never been compared and the expression of CD163 in the monocyte-dendritic cell lineage is not yet characterized. CD163 expression in dendritic cells (DCs) was investigated using multicolor flow cytometry in peripheral blood from 31 healthy donors and 15 HIV-1 patients in addition to umbilical cord blood from 5 newborn infants. Total RNA was isolated from MACS purified DCs and CD163 mRNA was determined with real-time reverse transcriptase polymerase chain reaction. The effect of glucocorticoid and phorbol ester stimulation on monocyte and dendritic cell CD163 and CD91 expression was investigated in cell culture of mononuclear cells using multicolor flow cytometry. We identified two CD163+ subsets in human blood with dendritic cell characteristics, CD163lo and CD163hi, together constituting a substantial fraction of DCs. Both subsets were characterized as [lin]- CD4+ ILT3+ HLA-DR+ CD11c+ by flow cytometry, and CD163 mRNA was readily detectable in MACS purified human DCs. CD163 on DCs was upregulated by glucocorticoid, and treatment by phorbol ester significantly decreased surface expression. Overall, the expression of CD163 on DCs was significantly increased in HIV-1 patients (19.3% [95% CI: 14.7-26.3%]) compared to healthy patients (10.5% [95% CI: 8.0-12.5]) p < 0.001. The CD163lo subset was CD16+, whereas the CD163hi subset was CD16-. Both subsets were CD91+, thereby constituting a subfraction of the recently described CD91+ CD11c+ dendritic cell subset. Coexpression of CD163 and CD91 was also demonstrated on human monocytes, which upon glucocorticoid treatment exhibited an increase in both CD163 and CD91 expression. We have now shown that CD163 and CD91 are coexpressed and coregulated on human monocytes. In addition, two subsets of CD163+ DCs constituting a fraction of the recently described CD91+ CD11c+ dendritic cell subset have been identified. The CD163 expression pattern suggests that if antigens are targeted to CD163 they may induce an immunostimulatory response like that of CD91-targeted antigens.

Antigens, CD↗

Spectral imaging and its applications in live cell microscopy.

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.

Animals↗

Sperm chromosome aneuploidy analysis in a man with globozoospermia.

OBJECTIVE: To determine the frequencies of chromosome aneuploidy and diploidy in sperm from a male with globozoospermia. DESIGN: Assessment of sperm chromosome aneuploidy and diploidy frequencies by multicolor fluorescence in situ hybridization (FISH) analysis. SETTINGS: University research laboratory. PATIENT(S): An infertile patient with round-headed sperm (globozoospermia). INTERVENTION(S): Sperm samples were obtained by masturbation for cytogenetic analysis. MAIN OUTCOME MEASURE(S): Aneuploidy and diploidy frequencies were assessed by multicolor FISH analysis for chromosomes 1, 15, 21, X, and Y and compared with those of five control donors. RESULT(S): A minimum of 10,000 sperm was analyzed per chromosome probe, for a total of 30,145 sperm. There was a statistically significantly increased frequency of XY disomy in the man with globozoospermia compared with the case in normal donors. The frequency of aneuploidy for chromosomes 1, 15, 21, XX, and YY was not statistically significantly increased. The frequency of diploidy was also not statistically significantly different. CONCLUSION(S): A previous report demonstrated an increased frequency of chromosome 15 aneuploidy in sperm of a globozoospermia patient who fathered a trisomy 15 conceptus. No other report has studied the frequency of chromosome 15 aneuploidy in these infertile men. Our study does not demonstrate an increased risk for chromosome 15 aneuploidy associated with globozoospermia. However, an elevated frequency of XY disomy was discovered, which is the most common type of chromosome abnormality observed in sperm of infertile men.

Adult↗

Use of the DsRed fluorescent reporter in zebrafish.

Green fluorescent protein (GFP) is firmly established as a fluorescent reporter for the imaging of specific tissues in zebrafish. The employment of other reporters such as DsRed in transgenic zebrafish has made multicolored labeling experiments possible. To date, several DsRed transgenic lines have been generated for lineage labeling, transplantation assays, and commercial applications. Advances in multicolored labeling experiments will depend on the implementation of newly engineered reporters and fusion proteins, as well as on innovative experiments that exploit the power of direct visualization.

Animals↗

Risk assessment for developing gliomas: a comparison of two cytogenetic approaches.

Chromosome instability (CIN) measured as chromosome aberrations has long been suggested as a cancer susceptibility biomarker. Conventional cytogenetic end-points are now being improved by combining molecular methods, which increases the sensitivity, specificity, and precision of the assay. In this study we examined both spontaneous and gamma-ray induced CIN in lymphocyte cultures from 51 previously untreated glioma patients and 51 age-, sex- and ethnicity-matched controls. CIN was assessed using two parallel methods: (1) the mutagen sensitivity (MS) assay and (2) the multicolor fluorescence in situ hybridization (FISH) assay. The frequency of spontaneous breaks was significantly higher in glioma patients (mean+/-S.D., 2.12+/-1.07) than in controls (1.24+/-0.86, P<0.001) when using the FISH assay but not the MS assay (0.019+/-0.02 and 0.019+/-0.01, respectively; P=0.915). Similarly, the frequency of induced chromatid breaks was significantly higher using the FISH assay (3.39+/-1.72) but not the MS assay (0.42+/-0.16) in the patients versus controls (2.08+/-1.18 and 0.37+/-0.15, respectively; P<0.001 and P=0.10, respectively). By using the median number of breaks in the controls as the cutoff value, we observed an odds ratio (ORs) of 5.13 (95% CI=2.23-12.1) for spontaneous and 4.86 (95% CI=2.08-11.4) for induced CIN using the FISH assay, whereas the ORs were 1.32 (95% CI=0.49-3.58) and 1.28 (95% CI=0.59-2.80) for spontaneous and induced CIN using the MS assay. There was also a significant increase in the frequency of hyperdiploid cells in the glioma cases which could only be detected using the FISH assay (OR=4.0, 95% CL=0.9-17.0). By combining both methods an estimated risk of 7.0 (95% CI=1.7-25.6) was observed. There was no correlation between the breaks detected by the two methods suggesting that each method is a measure of a different event. The results indicate that using the multicolor FISH assay for detection of CIN in peripheral blood lymphocytes in glioma patients is a more useful marker for risk assessment.

Adult↗

Chromosomal abnormalities in non-small cell lung carcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphase fluorescence in situ hybridization.

Human lung carcinogenesis is accompanied by complex chromosomal changes that may be detected in interphase cells by fluorescence in situ hybridization (FISH) assay using recently developed multitarget DNA probes. Touch preparations of 20 non-small cell lung carcinomas, sputum specimens from 3 patients with lung cancer and from 11 ex-smokers without lung cancer, and cultured benign bronchial epithelium of 42 high-risk smokers, 9 of whom had concurrent invasive carcinoma, were tested using a four-color FISH probe (LAVysion) targeting centromere 6, 5p15.2, 7p12 (EGFR), and 8q24 (MYC). Significantly high frequencies of abnormal cells were found in each of the 20 NSCLC (100%) and in the 3 sputum specimens from lung cancer patients. None of the cytologically normal sputa contained FISH abnormalities. Cultured bronchial epithelial cells from 11 of 42 patients (26%) were abnormal for at least one probe. Abnormal FISH patterns had no association with gender, presence of tumor or histology. Multicolor FISH can readily detect chromosomal abnormalities in imprints and sputa from lung carcinomas. Chromosomal aneusomy is also frequent in bronchial epithelial cells from long-term smokers. The prognostic significance of the multicolor LAVysion FISH probe set should be validated in a controlled clinical trial.

Bronchi↗

Theoretical study of the amazing firefly bioluminescence: the formation and structures of the light emitters.

Reaction mechanisms for the formation of the keto-form of oxyluciferin (OxyLH(2)) from the luciferin of fireflies via a dioxetanone intermediate are predicted using the B3LYP/6-31G theoretical method. The ring opening of a model dioxetanone and the decarboxylation proceed in one step via a singlet diradical transition structure with an activation barrier of 18.1 and an exothermicity of 90.8 kcal/mol. The S(0) --> S(1) vertical excitation energies predicted with time dependent density functional theory, TDDFT B3LYP/6-31+G, for the anionic and neutral forms of OxyLH(2) are in the range of 60 to 80 kcal/mol. These energetic results support the generally accepted theory of chemically initiated electron exchange luminescence (CIEEL). The chemical origin of the multicolor bioluminescence from OxyLH(2) is examined theoretically using the TDDFT B3LYP/6-31+G, ZINDO//B3LYP/6-31+G, and CIS/6-31G methods. A change in color of the light emission upon rotation of the two rings in the S(1) excited state of OxyLH(2) is unlikely because both possible emitters, the planar keto- and enol-forms, are minima on the S(1) potential energy surface. The participation of the enol-forms of OxyLH(2) in bioluminescence is plausible but not required to explain the multicolor emission. According to predictions at the TDDFT B3LYP level, the color of the bioluminescence depends on the polarization of the OxyLH(2) in the microenvironment of the enzyme-OxyLH(2) complex.

Animals↗

Genetics of human sperm.

PURPOSE: Chromosome abnormalities in sperm were studied by fluorescence in situ hybridization to determine the frequency and distribution of abnormalities in normal men and the effect of donor age on the frequency of abnormalities. Studies of chemotherapy and infertility patients assessed any increased risk in these populations. METHODS: Multicolor fluorescence in situ hybridization was performed on the sperm samples to assess aneuploidy frequencies for chromosomes 1, 2, 4, 9, 12, 13, 15, 16, 18, 20, 21, X, and Y as well as "sex ratios" and frequencies of diploid sperm. RESULTS: Most chromosomes yielded disomy estimates of approximately 0.1%, whereas the frequencies for chromosome 21 and the sex chromosomes were significantly elevated. The only chromosome to show a significant paternal age effect was YY disomy. Chemotherapy patients did not have an increased risk of aneuploid sperm 2-13 years after treatment. Infertility patients had an increased risk of disomy for chromosome 1, 13, 21, and XY. CONCLUSIONS: Multicolor fluorescence in situ hybridization analysis allows comparison of sperm from various populations of men and has demonstrated that infertile patients have a significant increase in the frequency of aneuploid sperm.

Adult↗

Molecular-cytogenetic characterization of the origin and the presence of pericentromeric euchromatin on minute supernumerary marker chromosomes (SMCs).

Small supernumerary marker chromosomes (SMCs) in human can be defined as additional centric chromosome fragments smaller than chromosome 20. For most small or minute SMCs a correlation with clinical symptoms is lacking, mostly due to problems in visualizing their euchromatic content. Recently we described two new molecular cytogenetic approaches for the comprehensive characterization of small SMCs, excluding those few cases with neo-centromeres. Minute SMCs, consisting preferentially of alpha-satellite DNA, are characterizable in one step by the centromere-specific multicolor FISH (cenM-FISH) approach. For further characterization of minute SMCs and eventually present euchromatic content, the recently developed centromere-near-specific multicolor FISH (subcenM-FISH) technique can be applied. These two approaches are highly informative and easy to perform, as demonstrated in the present report on the example of a prenatal case with a minute SMC derived from chromosome 3 cytogenetically described as min(3)(:p12.1 --> q11.2:).

Centromere↗

Long-term multiple color imaging of live cells using quantum dot bioconjugates.

Luminescent quantum dots (QDs)--semiconductor nanocrystals--are a promising alternative to organic dyes for fluorescence-based applications. We have developed procedures for using QDs to label live cells and have demonstrated their use for long-term multicolor imaging of live cells. The two approaches presented are (i) endocytic uptake of QDs and (ii) selective labeling of cell surface proteins with QDs conjugated to antibodies. Live cells labeled using these approaches were used for long-term multicolor imaging. The cells remained stably labeled for over a week as they grew and developed. These approaches should permit the simultaneous study of multiple cells over long periods of time as they proceed through growth and development.

Animals↗

Molecular cytogenetic detection of chromosomal breakpoints in T-cell receptor gene loci.

Chromosomal aberrations with breakpoints in T-cell receptor (TCR) gene loci are recurrent in several T-cell malignancies. Although the importance of interphase cytogenetics has been extensively shown in B-cell lymphomas, hardly any molecular cytogenetic tools are available for recurrent changes in T-cell disorders. Thus, we have established fluorescence in situ hybridization (FISH)-based break-apart assays for the TCRA/D (14q11), TCRB (7q34) and TCRG (7p14) genes and the TCL cluster (14q32). The assays were validated in normal controls as well as in 43 T-cell malignancies with cytogenetically proven 14q11, 7q34-35 or 7p13-21 aberrations. Breakpoints in TCRA/D, TCRB and TCRG could be diagnosed by these assays in 32/33 T-cell neoplasms with chromosome 14q11, 3/6 with 7q34-35 and 1/7 with 7p13-21 alterations, respectively. Application of the new FISH assays to a series of 24 angioimmunoblastic and 12 cutaneous T-cell lymphomas confirmed the cytogenetic evidence of lack of breakpoints in the TCRA/D or TCRB locus. Simultaneous detection of TCRA/D or TCRB breaks was achieved in a multicolor approach, which was further combined with detection of the T-cell-specific CD3 antigen in a multicolor FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasm) assay. These new FISH and FICTION assays provide sensitive, rapid and accurate tools for the diagnosis and biological characterization of T-cell malignancies.

Adolescent↗

A psychophysical dissection of the brain sites involved in color-generating comparisons.

We have used simple psychophysical methods to determine the sites of color-generating mechanisms in the brain. In our first experiment, subjects viewed an abstract multicolored "Mondrian" display through one eye and an isolated patch from the display through the other. With normal binocular/monocular viewing, the patch has a different color when viewed on its own (void mode) or as part of the Mondrian display (natural mode) [Land, E. H. (1974) Proc. R. Inst. G. B. 49, 23-58]. When the two stimuli were viewed dichoptically, with the patch occupying the position that it would occupy in the Mondrian complex under normal viewing, the patch always appeared in its void color. In a second experiment, when subjects viewed multicolored displays through a different narrow-band filter placed over each eye, the information from the two eyes was combined to result in new colors, which were not seen through either of the two eyes alone. Taken together, these results dissect color-generating mechanisms into two stages, located at different sites of the brain: The first occurs before the appearance of binocular neurons in the cortex and compares wavelength information across space, whereas the second occurs after the convergence of the input from the two eyes and synthetically combines the results of the first.

Adult↗

Mast cells in Waldenstrom's macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling.

Bone marrow (BM) mast cells (MC) are commonly found in association with lymphoplasmacytic cells (LPC) in patients with Waldenström's macroglobulinemia (WM). We therefore sought to clarify the role of MC in WM. Co-culture of sublethally irradiated HMC-1 MC, KU812 basophilic cells, or autologous BM MC along with BM LPC from WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. Furthermore, by immunohistochemistry, multicolor flow cytometry and/or RT-PCR analysis, CD40 ligand (CD154), a potent inducer of B-cell expansion, was expressed on BM MC from 32 of 34 (94%), 11 of 13 (85%), and 7 of 9 (78%) patients, respectively. In contrast, MC from five healthy donors did not express CD154. By multicolor flow cytometry, CD154 was expressed on BM LPC from 35 of 38 (92%) patients and functionality was confirmed by CD154 and CD40 agonistic antibody stimulation, which induced proliferation, support survival and/or pERK phosphorylation of LPC. Moreover, MC induced expansion of LPC from 3 of 5 patients was blocked in a dose dependent manner by use of a CD154 blocking protein. These studies demonstrate that in WM, MC may support tumor cell expansion through constitutive CD154-CD40 signaling and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.

B-Lymphocytes↗

Interpretation of low-level Plasmodium infection rates determined by ELISA for anophelines (Diptera: Culicidae) from Egyptian oases.

Plasmodium infection rates determined by enzyme-linked immunosorbent assay (ELISA) were compared for Anopheles sergentii (Theobald) and An. multicolor Cambouliu in Siwa Oasis, Egypt, an area with low-level Plasmodium vivax transmission, and in Bahariya and Farafra, two other Egyptian oases which appear to be free of malaria. Initial testing indicated that 4.4% (23 of 518) and 0.8% (4 of 518) of the An. sergentii were positive for P. vivax and P. falciparum, respectively, and that 1.4% (1 of 71) of the An. multicolor were positive for P. falciparum. However, after two confirmational tests, only 1.2% (6 of 518) of the An. sergentii remained consistently positive for P. vivax. Initial ELISA absorbance was not a useful predictor of potential false positive reactions in the P. vivax assay. Paradoxically, the six ELISA-positive An. sergentii were from the two malaria-free oases. This study raises the question of whether ELISA-positive reactions for anopheline vector species provides unequivocal evidence for transmission in areas of low malaria endemicity.

Animals↗