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PubMed2003

Rapid single-step FCGR3A genotyping based on SYBR Green I fluorescence in real-time multiplex allele-specific PCR.

A polymorphism at position 559 in the cDNA of the FCGR3A gene encoding the FcgammaRIIIa generates two allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158. This polymorphism is of major importance in immunopathology and in pharmacogenetics, especially monoclonal antibody treatments. In this study, we report a single-step and single-tube method for FCGR3A-158V/F genotyping by real-time multiplex allele-specific PCR and melting curve analysis in the presence of SYBR Green I fluorescent dye. Results obtained from 113 samples showed 100% accuracy compared to those obtained with a conventional PCR-based allele-specific restriction assay. Although this method requires expensive equipment, it is inexpensive in terms of consumables. It is also very rapid, reliable and suitable for large-scale analysis.

Alleles↗
PubMed2003

Multiplexed microsphere-based flow cytometric immunoassays for human cytokines.

Cytokines play a pivotal role in the regulation of immunologic, hematologic and wound-healing processes. They function to stimulate as well as inhibit the proliferation, differentiation and maturation of a variety of cell types. Thus, their functions are pleiotropic as well as interdependent to the extent that any cytokine may have effects that are synergistic or antagonistic with other cytokines. Cytokines also display redundancy when one mimics the functions of others. These characteristics imply that measuring the levels of one cytokine in a biologic system provides only a fraction of the information that is relevant to the existing physiologic state. A more realistic indication of the complexity of cellular interactions would include measurements of multiple cytokines at any time point. One method of multiplexed analysis can be performed by capture of the cytokines on an array of fluorescent microspheres for quantitation by flow cytometry. This technology has been applied to a variety of biomolecules, but simultaneous quantitation of multiple cytokines in a small sample volume has become rapid, inexpensive, reliable and informative.

Cytokines↗
PubMed2001

Th2 shift in mononeuritis multiplex and increase of Th2 cells in chronic inflammatory demyelinating polyneuropathy: an intracellular cytokine analysis.

To elucidate the T helper 1 (Th1)/T helper 2 (Th2) balance in various inflammatory neuropathies, we measured the ratio of intracellular interferon-gamma (IFN-gamma)-positive to IL-4-positive cells (intracellular IFN-gamma/IL-4 ratio) by flow cytometry in peripheral blood CD4(+) T cells of 14 patients with mononeuritis multiplex (MNM), 12 patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 10 patients with Guillain-Barré syndrome (GBS), 23 patients with neurodegenerative disorders and 36 healthy controls by intracellular labeling. The patients with MNM showed a significantly lower intracellular IFN-gamma/IL-4 ratio (P<0.05) and higher IL-4(+)/IFN-gamma(-) cell percentages (P<0.05) than the controls. The increase of IL-4(+)/IFN-gamma(-) cell percentages was especially prominent in MNM of unknown etiology (P<0.005). The patients with CIDP also showed significantly higher IL-4(+)/IFN-gamma(-) cell percentages (P<0.05) than the controls. The IL-4(+)/IFN-gamma(-) cell percentages were increased in some patients with GBS, but the difference was not significant compared with the controls. Thus, our results suggest that a Th2 shift is a characteristic of MNM and may play an important role in the development of the disease.

Adolescent↗
PubMed1999

Comparison of clinical and demographic features between affected pairs of Italian multiple sclerosis multiplex families; relation to tumour necrosis factor genomic polymorphisms.

We conducted a comparative analysis of clinical and demographic findings between pairs of relatives (36 sibling and 9 parent/child), concordant for Multiple Sclerosis (MS), from 40 MS Italian Multiplex families. A genetic TNF (alpha and beta) loci typing in 51 affected and 69 healthy relatives belonging to 25 of these families was also performed. The sib pairs resulted significantly concordant for age at onset (r=0.414, P<0.013), Progression Index (r=0.34, P<0.05) and sensory symptoms at onset (k=0.37), and significantly not concordant for sex (k=-0.37), whereas no concordance was found for year at onset and disease course. The only significant result in the small group of parent/child pairs was that parents developed MS at an age of 18.74 years significantly (P=0.020) greater than their children. Genomic analysis identified 13 variants of TNF-a alleles, 7 of TNF-b, 6 of TNF-d and 3 of TNF-e. No differences in the frequencies of the various TNF alleles were observed between affected and healthy relatives. The two-point lod-score analysis of the TNF locus showed not significant or negative results for the TNFalpha loci and slightly positive results (Zmax=0.4 at theta=0.2 cM) for the TNFbeta-b locus in the lowest penetrance dominant model. The Sib pair analysis, using combined TNFalpha and TNFbeta haplotypes, demonstrated a TNF allele sharing between affected sib-pairs which did not exceed the expected 50%. These results suggest that genetic factors may partially influence the disease onset and the progression rate in sibling pairs. A recall bias and/or an 'anticipation phenomenon' could explain the development of MS at an older age in parents than in their children. In this small-sized cohort of MS Italian families no significant associations were confirmed between TNF polymorphism and MS.

Adult↗
PubMed2002

Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR).

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

Animals↗
PubMed1986

Arthrogryposis multiplex congenita: spectrum of pathologic changes.

The pathologic features of muscle and/or spinal cord were studied in 96 infants and children with contractures of multiple joints (arthrogryposis multiplex congenita), usually in association with other congenital abnormalities. Ninety of these infants had a neurogenic form of arthrogryposis, and six had primary muscle disease. The neurogenic form, unlike the myopathic form, was usually associated with other congenital abnormalities. The most frequently associated congenital changes were low-set ears, micrognathia, wide flat nose, short neck, congenital heart disease, high-arched palate, hypoplastic lungs, and cryptorchidism. Some of the associated abnormalities could be attributed to muscle weakness, occurring during intrauterine development. A variety of skeletal muscle changes were observed, including primary myopathic alterations, fiber type predominance and disproportion, hypoplasia, aplasia, and denervation atrophy. When the primary alterations were in the spinal cord, abnormalities of anterior horn cells of several distinct types were recognized--absence of cells, diminution, dysgenesis, degeneration, and axonal reaction. The changes in anterior roots corresponded to those of the anterior horn cells.

Abnormalities, Multiple↗
PubMed1999

Two multiplex PCR-based DNA assays for the thrombosis risk factors prothrombin G20210A and coagulation factor V G1691A polymorphisms.

Two coagulation factor polymorphisms, G1691A in the factor V gene and G20210A in the prothrombin gene, are currently the most common known genetic risk factors for venous thrombosis in caucasian populations. Experimental conditions that permit simultaneous determination of these two polymorphisms using PCR-based DNA assays with the restriction enzyme, Hind III, were developed. Moreover, novel PCR-based DNA assays were established using primers that introduce specific cleavage sequences which then permit the simultaneous determination of these polymorphisms using Sac I restriction digestion analysis. Multiplex PCR-based DNA assays were used to analyze a family with a history of venous thrombosis in which these polymorphisms were both present and the results confirm the interesting variability of these genetic risk factors among family members who are symptomatic.

Biological Assay↗
PubMed2006

Development and implementation of multiplexed cell-based imaging assays.

Fluorescence microscopy, image analysis, and automated screening technologies are some of the most powerful tools enabling cell biologists to investigate complex signaling pathways and compound or siRNA effects on cellular function in individual cells. Researchers can now use multiple fluorescent probes to quantify effects on intracellular molecular events, measure phenotypic changes, and provide contextual information about cellular pathways not discernible by traditional single-parameter, end point experiments. This chapter focuses on fluorescent labeling techniques and methods for designing image-based assays, multiplexed readouts, and image analysis routines. Case studies are presented describing the use of cell-based imaging assays for monitoring cell proliferation, cell cycle stage, and apoptosis.

Animals↗
PubMed1995

Arthrogryposis multiplex congenita with maternal autoantibodies specific for a fetal antigen.

Fetal arthrogryposis multiplex congenita (AMC) is characterised by non-progressive multiple joint contractures, which may result in fetal death, and is heterogeneous in origin. It can associate with maternal myasthenia gravis and autoantibodies to muscle acetylcholine receptor (AChR). We found maternal antibodies that selectively inhibit the fetal form of the AChR in a mother who herself had no features of myasthenia gravis. Maternal autoantibodies specific for fetal antigens could be an unrecognised cause of other congenital disorders.

Adult↗
PubMed2001

Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization.

This study combines conventional cytogenetics, fluorescence in situ hybridization (FISH), multiplex-FISH and comparative genomic hybridization (CGH). In applying this multimodal approach on the human leukemia cell line K562, the chromosome composition was refined in detail and compared with data from the literature. A hypotriploid karyotype with a modal chromosome number of 67, and 21 unique marker chromosomes were identified. The classification of six markers was identical to published data and the composition of five further markers from the literature could be fully clarified for the first time. The composition of another five markers, which have been interpreted in divergent ways in different studies, were elucidated without doubt. Finally, five new markers of our study seem to have no equivalents in former studies, very likely due to limitations of conventional cytogenetics. The combinatory application of complementary techniques as shown in this study will be very useful to provide the basis of a refined genotype analysis on the chromosomal level.

Chromosome Mapping↗
PubMed2001

The use of multicolor fluorescence technologies in the characterization of prostate carcinoma cell lines: a comparison of multiplex fluorescence in situ hybridization and spectral karyotyping data.

Recent studies have identified several chromosome regions that are altered in primary prostate cancer and prostatic carcinoma cell lines. These targeted regions may harbor genes involved in tumor suppression. We used multiplex fluorescence in situ hybridization (M-FISH) to screen for genetic rearrangements in four prostate cancer cell lines, LNCaP, LNCaP.FCG, DU145, and PC3, and compared our results with those recently obtained using spectral karyotyping (SKY). A number of differences was noted between abnormalities characterized by SKY and M-FISH, suggesting variation in karyotype evolution and characterization by these two methodologies. M-FISH analysis showed that hormone-resistant cell lines (DU145 and PC3) contained many genetic alterations (> or =15 per cell), suggesting high levels of genetic instability in hormone-refractory prostate cancer. Most chromosome regions previously implicated in prostate cancer were altered in one or more of these cell lines. Several specific chromosome aberrations were also detected, including a del(4)(p14) and a del(6)(q21) in the hormone-insensitive cell lines, a t(1;15)(p?;q?) in LNCaP, LNCaP, and PC3, and a i(5p) in LNCaP.FCG, DU145, and PC3. These clonal chromosome abnormalities may pinpoint gene loci associated with prostate tumourigenesis, cancer progression, and hormone sensitivity.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2002

Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding.

We report the use of multiplex fluorescence in situ hybridization (M-FISH) to resolve chromosomal aberrations in seven established melanoma cell lines with hypotriploid to hypertetraploid complex karyotypes. By simultaneous identification of all human chromosomes in single FISH experiments using a set of 52 directly labeled, whole chromosome painting probes, cryptic chromosomal translocations and the origin of unclear chromosomal material in structural rearranged and marker chromosomes could be identified, refining the tumor karyotypes in all seven cell lines. The number of structural aberrations in each cell line assigned with combined M-FISH and DAPI banding analysis ranged from 15 to 45. Altogether, 275 breakpoints could be assigned to defined chromosomal regions or bands. The chromosome arms 1p, 6q, 7p, 9p, and 11q which are known to be nonrandomly associated with melanoma tumorigenesis, were frequently involved in chromosomal breaks and/or copy number changes. This study also demonstrated the practical usefulness of combining M-FISH with conventional cytogenetic banding techniques for the characterization of complex tumor karyotypes with massive genomic alterations.

Adult↗
PubMed2002

Application of multiplex fluorescence in situ hybridization in the cytogenetic analysis of primary gastric carcinoma.

The different genetic alterations observed in diffuse and intestinal types of gastric cancer suggest that these two pathological types may represent different disease entities. We present two cases of primary gastric carcinoma, a well-differentiated intestinal type adenocarcinoma and a poorly differentiated diffuse type adenocarcinoma, both studied by a 24-color multiplex fluorescence in situ hybridization technique (M-FISH). The well-differentiated intestinal type adenocarcinoma exhibited fewer structural abnormalities with five noncomplex translocations, deletions of chromosomes 5q, 6q, and 17q and an i(8q). In the case of poorly differentiated diffuse carcinoma, structural abnormalities predominated and normal homologues were mostly absent. But there were also similarities between the two cases: translocations on 1p and 9p; structural abnormalities of chromosome 8 with consistent loss of 8p; structural abnormalities of 12q; partial loss of chromosome 17 and 18; and polysomy of chromosome 20. This study shows that M-FISH is valuable in identifying hidden structural abnormalities and could, therefore, be useful in the investigation of primary solid tumors.

Adenocarcinoma↗
PubMed2002

Novel chromosome findings in bladder cancer cell lines detected with multiplex fluorescence in situ hybridization.

Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82, 253J, and SCaBER. Alterations of chromosome 9 are the most frequent cytogenetic and molecular findings in transitional cell carcinomas of all grades and stages, while changes of chromosomes 3, 4, 8, 9, 11, 14, and 17 are also frequently observed. In the present study, alterations previously described, including del(8)(p10), del(9)(p10), del(17)(p10), and overrepresentation of chromosome 20, as well as several novel findings, were observed. These novel findings were a del(15)(q15) and isochromosome 14q, both occurring in three of nine cell lines examined. These abnormalities may reflect changes in bladder tumor biology. M-FISH represents an effective preliminary screening tool for the characterization of complex tumor karyotypes.

Carcinoma, Squamous Cell↗
PubMed2004

Detection of genetic alterations in primary bladder carcinoma with dual-color and multiplex fluorescence in situ hybridization.

Cytogenetic studies of bladder cancer have shown several nonrandom aberrations. Numerical aberrations of both sex chromosomes were investigated in 32 primary bladder tumors with bicolor fluorescence in situ hybridization (FISH). Loss of chromosome Y and overrepresentation of chromosome X were observed in subgroups of male patients. Chromosome X was represented normally in female patients. Two of the above primary bladder tumors, a transitional cell carcinoma (TCC) and an adenocarcinoma, were further analyzed with both multiplex FISH (24-color M-FISH) and G-banding. Both cases exhibited 1) common breakpoints on 5q11 approximately q12 and 15q24; 2) involvement of the pericentromeric area of chromosome 13; 3) structural abnormalities of chromosomes 8 and 17, with loss of material on the short arm; 4) structural abnormalities involving chromosome 11; and 5) loss of chromosome Y. The TCC case also exhibited structural abnormalities of chromosome 9, resulting in loss of 9q. The combined G-banding and M-FISH findings could help reveal regions potentially involved in bladder tumorigenesis.

Carcinoma↗
PubMed2000

Multiplex fluorescence in situ hybridization and cross species color banding of a case of chronic myeloid leukemia in blastic crisis with a complex Philadelphia translocation.

Exciting new techniques in molecular cytogenetics--namely, spectral karyotyping, multiplex fluorescence in situ hybridization (M-FISH), and cross species color banding--have been recently developed. An increasing number of reports demonstrate the success of these procedures in providing additional cytogenetic information--identifying marker chromosomes and revealing the presence of previously undetected chromosomal changes. However, these procedures have their limitations, and their absolute sensitivity in the accurate identification of subtle chromosomal abnormalities remains to be established. M-FISH and color banding have been applied to a case of chronic myeloid leukemia with a complex Philadelphia translocation involving chromosomes 9, 17, and 22, which had initially been identified from G-banded chromosome analysis. The abnormalities were confirmed by chromosome "painting" and specific probes. Although M-FISH and color banding revealed no additional cryptic chromosomal changes, this study has clearly demonstrated the success of these multiple color FISH approaches in the accurate characterization of a complex rearrangement with subtle abnormalities.

Adolescent↗
PubMed2002

Antibody response to myelin oligodendrocyte glycoprotein and myelin basic protein depend on familial background and are partially associated with human leukocyte antigen alleles in multiplex families and sporadic multiple sclerosis.

We investigated the association of the antibody response to myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) with human leukocyte antigen (HLA) class II alleles in 41 patients with sporadic multiple sclerosis (MS) and 12 multiplex MS families. We found significantly increased antibody response to MOG and MBP in MS patients without any difference to asymptomatic relatives. HLA DRB1*04 was associated with IgM reactivity to MOG in MS patients, and DRB1*15 and DRB5 with anti-MOG IgA among asymptomatic relatives. We conclude that antibody responses to MOG and MBP depend on familial background. Moreover, the humoral immune reactivity against MOG is partially under control of certain HLA class II alleles.

Adult↗
PubMed2000

Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR.

A novel usage of multiplex reverse transcription polymerase chain reaction (m-RT-PCR) for simultaneous detection of multiple viruses is reported. By use of an oligo(dT), as a common primer, nearly full-length cDNAs can be synthesized. Furthermore, combining an oligo(dT) primer with a specific antisense primer can be used to simultaneously prime reverse transcription of both polyadenylated and non-polyadenylated RNAs. Four viral genera including five potato viruses [(carlavirus (PVS), polerovirus (PLRV), potexvirus (PVX), potyvirus (PVA and PVY))] and a viroid genus including a viroid genome (pospiviroid (PSTVd)) were used to develop various formats of m-RT-PCR. In artificially created viral RNA mixtures, all six RNA pathogens were detected successfully by uniplex- and m-RT-PCR. In naturally infected field grown tubers, m-RT-PCR detected infection of two to three viruses, which were present in the tubers.

DNA, Complementary↗