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Results for “Phenotype modifier”

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PubMed2001

Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems.

Metabolic profiling using gas chromatography-mass spectrometry technologies is a technique whose potential in the field of functional genomics is largely untapped. To demonstrate the general usefulness of this technique, we applied to diverse plant genotypes a recently developed profiling protocol that allows detection of a wide range of hydrophilic metabolites within a single chromatographic run. For this purpose, we chose four independent potato genotypes characterized by modifications in sucrose metabolism. Using data-mining tools, including hierarchical cluster analysis and principle component analysis, we were able to assign clusters to the individual plant systems and to determine relative distances between these clusters. Extraction analysis allowed identification of the most important components of these clusters. Furthermore, correlation analysis revealed close linkages between a broad spectrum of metabolites. In a second, complementary approach, we subjected wild-type potato tissue to environmental manipulations. The metabolic profiles from these experiments were compared with the data sets obtained for the transgenic systems, thus illustrating the potential of metabolic profiling in assessing how a genetic modification can be phenocopied by environmental conditions. In summary, these data demonstrate the use of metabolic profiling in conjunction with data-mining tools as a technique for the comprehensive characterization of a plant genotype.

Cluster Analysis↗

PubMed2006

Automated local bright feature image analysis of nuclear protein distribution identifies changes in tissue phenotype.

The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently stained nuclear protein NuMA in different mammary phenotypes obtained using 3D cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from 3D confocal images. Prominent features of fluorescently stained NuMA were detected by using a previously undescribed local bright feature analysis technique, and their normalized spatial density was calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features when nonneoplastic cells underwent phenotypically normal acinar morphogenesis. Conversely, we did not detect any reorganization of NuMA during formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating nonneoplastic from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues.

Antigens, Nuclear↗

PubMed2003

HLA-A and -B alleles and haplotypes in 240 index patients with common variable immunodeficiency and selective IgG subclass deficiency in central Alabama.

BACKGROUND: We wanted to quantify HLA-A and -B phenotype and haplotype frequencies in Alabama index patients with common variable immunodeficiency (CVID) and selective IgG subclass deficiency (IgGSD), and in control subjects. METHODS: Phenotypes were detected using DNA-based typing (index cases) and microlymphocytotoxicity typing (controls). RESULTS: A and B phenotypes were determined in 240 index cases (114 CVID, 126 IgGSD) and 1,321 controls and haplotypes in 195 index cases and 751 controls. Phenotyping revealed that the "uncorrected" frequencies of A*24, B*14, B*15, B*35, B*40, B*49, and B*50 were significantly greater in index cases, and frequencies of B*35, B*58, B*62 were significantly lower in index cases. After Bonferroni corrections, the frequencies of phenotypes A*24, B*14, and B*40 were significantly greater in index cases, and the frequency of B*62 was significantly lower in index cases. The most common haplotypes in index cases were A*02-B*44 (frequency 0.1385), A*01-B*08 (frequency 0.1308), and A*03-B*07 (frequency 0.1000), and the frequency of each was significantly greater in index cases than in control subjects ("uncorrected" values of p < 0.0001, 0.0252, and 0.0011, respectively). After performing Bonferroni corrections, however, the frequency of A*02-B*44 alone was significantly increased in probands (p < 0.0085). Three other haplotypes were also significantly more frequent in index cases (A*03-B*14, A*31-B*40, and A*32-B*14). The combined frequencies of three latter haplotypes in index patients and control subjects were 0.0411 and 0.0126, respectively ("uncorrected" value of p < 0.0002; "corrected" value of p = 0.0166). Most phenotype and haplotype frequencies in CVID and IgGSD were similar. 26.7% of index patients were HLA-haploidentical with one or more other index patients. We diagnosed CVID or IgGSD in first-degree or other relatives of 26 of 195 index patients for whom HLA-A and -B haplotypes had been ascertained; A*01-B*08, A*02-B*44, and A*29-B*44 were most frequently associated with CVID or IgGSD in these families. We conservatively estimated the combined population frequency of CVID and IgGSD to be 0.0092 in adults, based on the occurrence of CVID and IgGSD in spouses of the index cases. CONCLUSIONS: CVID and IgGSD in adults are significantly associated with several HLA haplotypes, many of which are also common in the Alabama Caucasian population. Immunoglobulin phenotype variability demonstrated in index cases and family studies herein suggests that there are multiple gene(s) on Ch6p or other chromosomes that modify immunoglobulin phenotypes of CVID and IgGSD. The estimated prevalence of CVID and IgGSD in central Alabama could be reasonably attributed to the fact that many HLA haplotypes significantly associated with these disorders are also common in the general population.

Adult↗

PubMed1996

Variants at the secretory phospholipase A2 (PLA2G2A) locus: analysis of associations with familial adenomatous polyposis and sporadic colorectal tumours.

The Min mouse is a model for human familial adenomatous polyposis (FAP), an autosomal dominant disease characterised by multiple adenomatous gastrointestinal polyps. The severity of the Min phenotype is modified by a locus (Mom1) on mouse chromosome 4, at a position syntenic with human chromosome 1p35-p36. The secretory phospholipase A2 (Pla2s) gene is a candidate for this modifier locus and there is evidence that a locus on human chromosome 1p35-p36 acts to modify the severity of human duodenal FAP. We have analysed the human secretory phospholipase A2 locus (PLA2G2A) for variants that could directly influence the FAP phenotype. We found no PLA2G2A variants predicted to result in functional variation in the phospholipase A2 protein. Two PLA2G2A polymorphisms were, however, discovered, one a 'silent' base change in exon 3 and another in a noncoding region. Three other variants (possible mutations) were found in non-coding regions. In 70 FAP patients from 20 families, no associations were found between the severity of duodenal polyposis and any PLA2G2A variant. One allele at the exon 3 polymorphic site did, however, occur more often then expected in patients with relatively severe colonic FAP. Although of borderline statistical significance, this association, if genuine, is likely to result from linkage disequilibrium between the PLA2G2A alleles studied and undetected genetic variation at a closely linked locus. The frequency of the alleles at both polymorphic sites has also been determined in the germ line of patients with sporadic colorectal adenomas and carcinomas and in random controls, but no differences were found among these groups. Our results suggest that PLA2G2A variants do not influence inherited or sporadic colonic tumours. A linked locus may be a modifier of human FAP, but does not influence the risk of colorectal tumours in the general population.

Adenomatous Polyposis Coli↗

PubMed1998

Relationship between genotype and phenotype. Thalassemia intermedia.

Thalassemia intermedia encompasses a number of clinical conditions ranging in severity from beta-thalassemia carrier state to transfusion-dependent thalassemia major. The molecular bases of thalassemia intermedia, only partially defined, are very heterogeneous, but in general any factor able to reduce the globin-chain imbalance results in a milder form of thalassemia. These factors are the presence of a silent or mild beta-thalassemia allele, associated with a high residual beta-globin production, and the coinheritance of alpha-thalassemia or of genetic determinants that increase the gamma-chain production. Less frequent mechanisms are double heterozygosity for beta-thalassemia and triplicated alpha genes, and the presence of a hyperunstable hemoglobin variant. However, for a consistent number of beta zero-thalassemia homozygotes with a thalassemia intermedia phenotype the modifying factor has not been defined yet. In contrast, there are simple beta-thalassemia carriers who, for unknown reasons, have an unusually severe clinical phenotype.

Blood Transfusion↗

PubMed1996

Discordant phenotype in siblings with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a congenital humoral immunodeficiency caused by a defect in a B-cell-specific signaling molecule, Btk. There has been little concordance of phenotype with genotype in this disorder, and defects in Btk cause immunodeficiencies that range from mild impairment to complete inability to produce antibodies. The factors modifying the phenotype of XLA are not understood. The current study is the first description of two male siblings with identical T134 --> C mutations in the translation initiation ATG of Btk who have different clinical phenotypes. The proband lacks immunoglobulins and B cells and has recurrent infections, while the elder, affected brother has normal levels of IgG and IgM and very few infections. Both have undetectable levels of Btk kinase activity in circulating mononuclear cells. Complete sequencing of Btk gene transcripts in both brothers revealed no additional mutations to account for the discordant phenotypes. This description provides unequivocal evidence that the phenotype of XLA is influenced by factors additional to the Btk gene.

Adolescent↗

PubMed1998

Lipoprotein (a) concentration and apolipoprotein (a) phenotype in subjects with type 2 diabetes mellitus.

OBJECTIVE: To investigate the apolipoprotein (a) [apo(a)] polymorphism in the patients with type 2 diabetes mellitus and its relationship with complications. METHODS: In this study, we tested apo(a) phenotype via modified Utermann and Guo method in the 40 non-diabetic controls and 176 subjects with type 2 diabetes and analyzed the relationship between apo(a) phenotypes and micro- and macrovascular complications, including nephropathy, neuropathy, retinopathy, hypertension, coronary heart disease and cerebral infarction. RESULTS: Among the 40 non-diabetic controls, the frequencies of S3S2, S4 and S4S2 were 20%, 70% and 10% respectively and the serum lipoprotein (a) [Lp(a)] level was 0.08 +/- 0.07 mg/L. While the frequencies of S2, S3, S3S2, S4, S4S2, S4S3 were 18.18%, 20.45%, 17.05%, 34.09%, 4.55% and 5.68% in the diabetics and the Lp(a) concentration was 0.13 +/- 0.11 mg/L, with significant difference between the diabetics and non-diabetic controls. In comparison with the diabetics without complications, the frequency of apo(a) phenotype significantly differed in patients with nephropathy, nephropathy, hypertension, coronary heart disease and cerebral infarction except for diabetic retinopathy. In comparison with patients with S2 phenotype, the levels of fasting plasma glucose (FPG), 2-hour postprandial plasma glucose (2hPG) and glycated hemoglobin A1c (HbA1c) were lower in patients with S4 phenotype. The concentration of Lp(a) and urine albumin index (Alb/Cr) were significantly different among diabetics with different apo(a) phenotype, the highest being in patients with S2, secondly S3, and the lowest S4. CONCLUSION: There were significant differences in the frequency of apo(a) phenotype between subjects with type 2 diabetes and non-diabetic controls, and also in diabetics with or without microvascular and macrovascular diseases. The underlying linkage might be microalbuminuria and insulin resistance.

Adult↗

PubMed1988

Effect of methyl methanesulfonate on type 5 adenovirus DNA integration and the phenotypic properties of cold-sensitive type 5 adenovirus-transformed cloned rat embryo fibroblast cells.

Pretreatment of a cloned rat embryo fibroblast cell line (CREF) with methyl methanesulfonate (MMS) prior to infection with a specific cold-sensitive type 5 adenovirus mutant, H5hr1, results in a unique carcinogen enhancement of transformation phenotype. MMS induces a dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for carcinogen-induced cell toxicity. To determine if the carcinogen enhancement of transformation phenotype was a consequence of the carcinogen altering the pattern of type 5 adenovirus (Ad5) DNA integration into the genome of CREF cells and/or if carcinogen treatment modified the phenotype of established H5hr1-transformed CREF cells, we have analyzed a series of single cell-derived H5hr1-transformed CREF cultures which were isolated from cultures pretreated with carcinogen-solvent or MMS prior to infection with H5hr1. Analysis of viral DNA integration by DNA filter-transfer hybridization (Southern blotting) indicated that MMS pretreatment did not increase the copy number of Ad5 DNA sequences which persisted in H5hr1-transformed clones or result in transformants which contained identical DNA restriction enzyme cleavage patterns. MMS-pretreated H5hr1-transformed clones also did not differ significantly from solvent-pretreated H5hr1-transformed clones in their ability to grow in agar, bind 125I-epidermal growth factor, or form tumors in athymic nude mice. MMS-pretreated H5hr1-transformed CREF clones retained a similar cold-sensitive negative regulation in the expression of the transformed cell phenotype as did H5hr1-transformed clones not exposed to carcinogens. These findings suggest that the unique carcinogen enhancement of transformation phenotype displayed by CREF cells pretreated with MMS prior to infection with H5hr1 does not result in transformants which either contain increased concentrations of Ad5 DNA or similar patterns of Ad5 DNA integration. Furthermore, carcinogen-pretreated H5hr1 transformants did not display novel phenotypes not expressed by cloned H5hr1-transformed CREF cell lines exposed to solvent prior to viral infection.

Adenoviruses, Human↗

PubMed2007

Immunization of LDL receptor-deficient mice with beta2-glycoprotein 1 or human serum albumin induces a more inflammatory phenotype in atherosclerotic plaques.

Antiphospholipid antibodies are a risk factor for venous and arterial thrombosis and may contribute to the development of atherosclerosis.The aim of this study was to investigate whether antibodies to human beta2-glycoprotein 1 (beta2GP1), as a model of antiphospholipid antibodies, modify the phenotype of atherosclerotic lesions. LDL receptor-deficient mice were immunized with human beta2GP1, human serum albumin (HSA), or not immunized, and fed a high-cholesterol diet for 14 weeks. Some mice also received pravastatin. Immunization with human beta2GP1 or HSA resulted in formation of autoantibodies recognizing murine beta2GP1 or murine albumin, respectively. We quantified atherosclerotic lesion development and mRNA levels of inflammation associated proteins in the thoraco-abdominal aorta as well as lesion development,cellular composition and collagen content in the aortic roots. Immunization with beta2GP1 or HSA had no effect on lesion size,but modified the expression in plaque areas of several inflammation-associated proteins. Expression of matrix metalloproteinase-9, tissue factor, interferon-gamma and CD25 was highest in the thoraco-abdominal aorta of beta2GP1-immunized mice, lowest in non-immunized mice and intermediate in HSA-immunized animals. Immunization with beta2GP1, but not HSA, resulted in a lower smooth muscle cell and collagen content of lesions in aortic roots. Statin treatment partially reversed the effects of beta2GP1 immunization. We conclude that immunization with beta2GP1, and to a lesser extent with HSA, leads to modifications in the cellular and protein composition of atherosclerotic plaques, which are associated with a more inflammatory phenotype. Statin treatment partially prevents these changes.

Animals↗

PubMed1999

Clinical studies of families with hearing loss attributable to mutations in the connexin 26 gene (GJB2/DFNB1)

OBJECTIVE: This retrospective study describes the phenotype associated with the single most common cause of genetic hearing loss. The frequency of childhood deafness is estimated at 1/500. Half of this hearing loss is genetic and approximately 80% of genetic hearing loss is nonsyndromic and inherited in an autosomal recessive manner. Approximately 50% of childhood nonsyndromic recessive hearing loss is caused by mutations in the connexin 26 (Cx26) gene (GJB2/DFNB1), making it the most common form of autosomal recessive nonsyndromic hearing loss with a carrier rate estimated to be as high as 2.8%. One mutation, 35delG, accounts for approximately 75% to 80% of mutations at this gene. METHODS: Hearing loss was examined in 46 individuals from 24 families who were either homozygous or compound heterozygous for Cx26 mutations. A subset of these individuals were examined for vestibular function, otoacoustic emissions, auditory brainstem response, temporal bone computed tomography, electrocardiography, urinalyses, dysmorphology, and thyroid function. RESULTS: Although all persons had hearing impairment, no consistent audiologic phenotype was observed. Hearing loss varied from mild-moderate to profound, even within the group of families homozygous for the common mutation 35delG, suggesting that other factors modify the phenotypic effects of mutations in Cx26. Furthermore, the hearing loss was observed to be progressive in a number of cases. No associations with inner ear abnormality, thyroid dysfunction, heart conduction defect, urinalyses, dysmorphic features, or retinal abnormality were noted. CONCLUSION: Newborns with confirmed hearing loss should have Cx26 testing. Cx26 testing will help define a group in which approximately 60% will have profound or severe-profound hearing loss and require aggressive language intervention (many of these patients will be candidates for cochlear implants).

Child↗

PubMed2004

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero.

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ss-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.

Animals↗

PubMed2004

The recently identified type 2A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype.

The most common form of hereditary haemochromatosis is an adult-onset condition usually associated with the HFE C282Y/C282Y genotype. The phenotypic expression of this genotype is heterogeneous and depends on a complex interplay of genetic and non-genetic factors. The aim of the present study was to determine if mutations in the recently identified HJV gene were associated with more severe iron overload phenotypes in C282Y homozygous patients. From a cohort of 310 C282Y homozygous patients, we found nine (six males and three females) with an additional HJV missense mutation in the heterozygous state (S105L, E302K, N372D, R335Q or the previously described L101P and G320V). The iron indices of eight patients appeared to be more severe than those observed in C282Y homozygous patients of identical sex and similar age ranges. The mean serum ferritin concentration of the six males with an HJV mutation was significantly higher than that of C282Y homozygous males without an additional mutation [2350.3 (sigma=1429.9) versus 1227.2 (sigma=1130.1) microg/l; P=0.0233, Student's t-test]. We have recently reported that mutations in the gene that encodes hepcidin (HAMP) could explain one part of the C282Y/C282Y-related phenotypic heterogeneity by accentuating the iron burden. Our new data reveal that mutations in the HJV gene could be associated with a similar effect. Taken together, these results emphasize that a search for modifier genes could enable us to more precisely distinguish those C282Y homozygous patients with a higher risk to develop a severe iron overload and, consequently, clinical complications.

DNA Mutational Analysis↗

PubMed2001

Current perspectives on BRCA1- and BRCA2-associated breast cancers.

The identification of two breast cancer predisposition genes, BRCA1 and BRCA2, in the mid-1990s has led to a better understanding of the molecular pathogenesis of hereditary breast cancer and to a new era in breast cancer research. The present article reviews the current state of knowledge regarding the biology of BRCA1 and BRCA2, the cancer risks associated with carrying a pathogenic mutation in either of these genes and the possible genetic and environmental risk modifiers. The phenotypes of BRCA1- and BRCA2-associated hereditary breast cancers are reviewed. Research into BRCA1- and BRCA2-associated breast cancer is in its infancy and much remains to be learned, particularly about modifiers of genetic risk and the clinical implications of carrying a mutation in one of these two genes. Australia has an excellent research infrastructure in place, through the Australian Breast Cancer Family Study and the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, to contribute substantially to future research in this area.

Animals↗

PubMed1994

Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila.

Closely linked repeats of a Drosophila P transposon carrying a white transgene were found to cause white variegation. Arrays of three or more transgenes produced phenotypes similar to classical heterochromatin-induced position-effect variegation (PEV), and these phenotypes were modified by known modifiers of PEV. This effect on the repeated transgenes was much stronger for a site near centric heterochromatin than it was for a medial site, and it strengthened with increasing copy number. Differences between variegated phenotypes could be accounted for if different topological structures were generated by pairing between closely linked repeat sequences. We propose that pairing of repeats underlies heterochromatin formation and is responsible for diverse gene silencing phenomena in animals and plants.

Animals↗

PubMed1979

Mitochondrial and nuclear mutations that affect the biogenesis of the mitochondrial ribosomes of yeast. I. Genetics.

We have isolated about five hundred temperature-sensitive mutants specific for the mitochondrial functions. Their growth on glycerol is defective at 36 degrees C and/or 20 degrees C. While most of the mutations were nuclearly inherited, about thirty were found to be of mitochondrial origin. 1) Four mitochondrial mutations (three cryosensitive, one thermosensitive) were localized close to chloramphenicol and erythromycin resistance loci of the mitochondrial DNA, that is in the region coding for the 23 S ribosomal RNA. One of the mutation interfered with the expression of the chloramphenicol resistance gene. 2) A dozen nuclear mutations were isolated from a strain which is labelled with mitochondrial drug resistance markers (chloramphenicol, erythromycin, and paromomycin). Among the temperature sensitive respiratory deficient mutants, we have selected the mutations that supress the resistant phenotypes. We describe two non allelic such mutations, one being cryosensitive, the other thermosensitive. Both supress the expression of the mitochondrial chloramphenicol resistance gene. The temperature sensitive growth on glycerol and the modified antibiotic phenotype segregated together as a single recessive mutation. A biochemical study of these mutants is presented in a joint paper, confirming their presumed ribosomal nature.

Cell Nucleus↗

PubMed2005

Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.

Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, we are using a genetic approach in Drosophila. Here, we show that Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 (GstS1) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD.

Animals↗

PubMed1969

The relation between peroxidase activity and the morphological expression of the hooded gene in barley.

Reproductive meristems of the hooded (KK) genotype of barley at the stage of young lemma primordia were injected with the inhibitors sodium azide, hydrazine, and hydroxylamine at concentrations of 10(-3), 10(-4), and 10(-5)M, as well as with phenylboric acid. These treatments all brought about an altered expression of the adult phenotype in the direction of awned (kk) genotype. Analysis of tissues treated with the same reagents revealed a reduction in peroxidase activity which was proportional to the effect of each inhibitor in modifying the phenotypic expression of the gene. Exogenous IAA injected in the same manner brought about a similar modification of the phenotype and parallel reduction of peroxidase activity, but to a lesser degree. These results are believed to be caused by modifications of the action of the IAA oxidase system in the developing meristems.

Journal Article↗

PubMed2002

Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells.

Leukemic T cell lines have facilitated signal transduction studies but their physiological relevance is restricted. The use of primary T lymphocytes overcomes this limitation but it has long been speculated that methodological aspects of blood collection and the isolation procedure modify the phenotype of the cell. Here we demonstrate that several characteristics of human peripheral T cells are affected by the selection conditions. A significantly higher induction of the chemokine receptor CXCR4 was observed on CD4+ lymphocytes isolated by sheep red blood cell (SRBC) rosetting and CD4 MicroBeads as compared with positively selected CD4+ cells where the antibody/bead complex was immediately detached. These latter cells expressed CXCR4 at levels equivalent to that observed on CD4+ lymphocytes obtained by negative antibody-mediated selection. Furthermore, CD4+ cells isolated by SRBC rosetting and CD4 MicroBeads formed aggregates upon in vitro culture. CD4+ lymphocytes obtained by SRBC rosetting as well as those isolated following positive selection demonstrated basal phosphorylation of the extracellular signal-regulated kinase (ERK)-2. Altogether these data suggest that certain discrepancies concerning signal transduction in primary human T cells can be attributed to the selection conditions. Thus, it is essential to establish the parameters influenced by the isolation protocol in order to fully interpret T cell responses to antigens, chemokines, and cytokines.

Blotting, Western↗