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At least 1,045 records · Page 58Linked to original sources
PubMed2004

Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis.

OBJECTIVE: To determine novel genes regulated by tumor necrosis factor alpha (TNFalpha) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIkappaB-dominant negative (AdIkappaB-DN) (n = 3) or with control AdTet expressing the reverse tetracycline trans-activator (n = 3). The cells were stimulated for 3 hours with TNFalpha, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarray-identified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. RESULTS: Blocking of NF-kappaB by AdIkappaB-DN was associated with a down-modulation of antiapoptosis genes, including BIRC-3, and several novel genes, including GG2-1, a TNFalpha-inducible FLIP-like gene. Other families of genes that were significantly down-regulated by AdIkappaB-DN included cytokines/chemokines (interleukin-1beta [IL-1beta], IL-8, IL-15, and RANTES), adhesion molecule (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), and unique genes that have not previously been reported to be regulated by TNFalpha in RA. Inhibition of the GG2-1 gene using the siRNA technique resulted in significantly enhanced apoptosis, decreased proliferation, and decreased production of MMP-1 in TNFalpha-stimulated RASFs. CONCLUSION: These studies provide a comprehensive analysis of genes that are differentially regulated by TNFalpha signaling and NF-kappaB nuclear translocation in RASFs and demonstrate methods for confirming the expression and functional significance of such genes.

Apoptosis↗
PubMed2004

The V(lambda)-J(lambda) repertoire of patients with systemic lupus erythematosus manifests characteristics of the natural antibody repertoire.

OBJECTIVE: To understand in detail the mechanisms of autoantibody production in patients with systemic lupus erythematosus (SLE), we performed a comprehensive analysis of the normal human immunoglobulin light chain V(lambda) repertoire and compared it with the V(lambda) repertoire in SLE patients. METHODS: The SLE V(lambda) repertoire of B cells obtained from 3 SLE patients was analyzed and compared in detail with the V(lambda) repertoire of IgM+ B cells obtained from 3 human fetal spleens and IgM+,CD5+ B cells obtained from 2 normal adults. Conventional IgM+,CD5- B cells obtained from normal adults were used as controls. V(lambda)-J(lambda) rearrangements were amplified from the genomic DNA of individual B cells by polymerase chain reaction. RESULTS: The expressed V(lambda) repertoire of SLE patients contained several similarities with the expressed repertoire of the fetus and the adult CD5+ B cells. The V(lambda) genes 3L and 1G were overexpressed in the fetus, the adult CD5+ B cells, and the patients with SLE. The selection for rearrangements with restricted junctional diversity by utilization of homology-mediated joining, together with diminished N nucleotide addition, was a prominent feature of fetal, adult CD5+, and SLE B cell repertoires. Furthermore, profound expansion of V(lambda) clones with identical third complementarity-determining regions was observed in the adult CD5+, fetal, and SLE B cell repertoires. Notably, significant numbers of expanded adult CD5+ B cells, fetal, and SLE V(lambda) clones utilized homology-mediated joining at the V(lambda)-J(lambda) junctions. CONCLUSION: These data demonstrate that the SLE V(lambda)-J(lambda) repertoire manifests characteristics of normal adult IgM+,CD5+ and fetal B cell populations that are known to be enriched for the production of natural autoantibodies.

Adult↗
PubMed2005

Ultraviolet radiation-induced injury, chemokines, and leukocyte recruitment: An amplification cycle triggering cutaneous lupus erythematosus.

OBJECTIVE: To investigate the activation and recruitment pathways of relevant leukocyte subsets during the initiation and amplification of cutaneous lupus erythematosus (LE). METHODS: Quantitative real-time polymerase chain reaction was used to perform a comprehensive analysis of all known chemokines and their receptors in cutaneous LE lesions, and the cellular origin of these chemokines and receptors was determined using immunohistochemistry. Furthermore, cytokine- and ultraviolet (UV) light-mediated activation pathways of relevant chemokines were investigated in vitro and in vivo. RESULTS: In the present study, we identified the CXCR3 ligands CXCL9 (interferon-gamma [IFNgamma]-induced monokine), CXCL10 (IFNgamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant) as being the most abundantly expressed chemokine family members in cutaneous LE. Expression of these ligands corresponded with the presence of a marked inflammatory infiltrate consisting of mainly CXCR3-expressing cells, including skin-homing lymphocytes and blood dendritic cell antigen 2-positive plasmacytoid dendritic cells (PDCs). Within cutaneous LE lesions, PDCs accumulated within the dermis and were activated to produce type I IFN, as detected by the expression of the IFNalpha-inducible genes IRF7 and MxA. IFNalpha, in turn, was a potent and rapid inducer of CXCR3 ligands in cellular constituents of the skin. Furthermore, we demonstrated that the inflammatory CXCR3 ligands cooperate with the homeostatic chemokine CXCL12 (stromal cell-derived factor 1) during the recruitment of pathogenically relevant leukocyte subsets. Moreover, we showed that UVB irradiation induces the release of CCL27 (cutaneous T cell-attracting chemokine) from epidermal compartments into dermal compartments and up-regulates the expression of a distinct set of chemokines in keratinocytes. CONCLUSION: Taken together, our data suggest an amplification cycle in which UV light-induced injury induces apoptosis, necrosis, and chemokine production. These mechanisms, in turn, mediate the recruitment and activation of autoimmune T cells and IFNalpha-producing PDCs, which subsequently release more effector cytokines, thus amplifying chemokine production and leukocyte recruitment, finally leading to the development of a cutaneous LE phenotype.

Cells, Cultured↗
PubMed2006

Impaired recognition of apoptotic neutrophils by the C1q/calreticulin and CD91 pathway in systemic lupus erythematosus.

OBJECTIVE: A deficiency in a subcomponent of C1q can result in increased susceptibility to autoimmune diseases such as systemic lupus erythematosus (SLE). The monocyte endocytic receptor CD91 is implicated in the endocytosis of apoptotic neutrophils via interactions with C1q and calreticulin. In this clinical study, we studied the binding of C1q to leukocytes and determined whether C1q bound specifically to calreticulin and CD91 on cells undergoing apoptosis in SLE. METHODS: Proximal antibody phage display, calreticulin-transfected cells, and immunocytochemical and confocal techniques were used in a comprehensive analysis of direct binding of C1q to apoptotic neutrophils that were obtained from healthy individuals and from patients with SLE. In addition, apoptotic cellular systems were assessed in vitro. RESULTS: C1q appeared to colocalize to apoptotic blebs on the surface of leukocytes in association with both calreticulin and CD91, as determined by phage display and transfected cell studies. However, C1q did not bind to apoptotic cells isolated from SLE patients, despite the positivity of the cells for both calreticulin and CD91. Surface expression of calreticulin decreased on neutrophils as they aged, but increased on monocytes. In an apoptotic phagocytic assay, the addition of C1q and calreticulin significantly enhanced the phagocytosis of apoptotic cell debris by monocyte-derived cells. CONCLUSION: These observations indicate that neutrophils from SLE patients have a reduced ability to be recognized and removed by the C1q/calreticulin/CD91-mediated apoptotic pathway, despite the presence of main apoptotic recognition partners. This suggests that an additional component, as yet unidentified, acts as a C1q binding partner on apoptotic cells, and this component may be lacking in cells isolated from SLE patients.

Adolescent↗
PubMed2006

Expression profiling of transforming growth factor beta superfamily genes in developing orofacial tissue.

BACKGROUND: Numerous signaling molecules have been shown to participate in the dynamic process of orofacial development. Among these signal mediators, members of the transforming growth factor beta (TGFbeta) superfamily have been shown to play critical roles. Developing orofacial tissue expresses TGFbeta and bone morphogenetic protein (BMP) mRNAs, their protein isoforms and TGFbeta- and BMP-specific receptors. All these molecules display unique temporospatial patterns of expression in embryonic orofacial tissue, suggesting functional roles in orofacial development. For example, the TGFbetas and BMPs regulate maxillary mesenchymal cell proliferation and extracellular matrix synthesis. This is particularly noteworthy in that perturbation of either process results in orofacial clefting. Although the cellular and phenotypic effects of the TGFbeta superfamily of growth factors on embryonic orofacial tissue have been extensively studied, the specific genes that function as effectors of these cytokines in orofacial development have not been well defined. METHODS: In the present study, oligonucleotide-based microarray technology was utilized to provide a comprehensive analysis of the expression of the panoply of genes related to the TGFbeta superfamily, as well as those encoding diverse groups of proteins functionally associated with this superfamily, during orofacial ontogenesis. RESULTS: Of the 7000 genes whose expression was detected in the developing orofacial region, 249 have been identified that encode proteins related to the TGFbeta superfamily. Expression of some (27) of these genes was temporally regulated. In addition, several candidate genes, whose precise role in orofacial development is still unknown, were also identified. Examples of genes constituting this cluster include: TGFbeta1-induced antiapoptotic factor-1 and -2, TGFbeta-induced factor 2, TGFbeta1 induced transcript-1 and -4, TGFbeta-inducible early growth response 1, follistatin-like 1, follistatin-like 3, transmembrane protein with EGF-like and two follistatin-like domains (Tmeff)-1 and -2, nodal modulator 1, various isoforms of signal transducers and activators of transcription (Stat), notch, and growth and differentiation factors. CONCLUSIONS: Elucidation of the precise physiological roles of these proteins in orofacial ontogenesis should provide unique insights into the intricacies of the TGFbeta superfamily signal transduction pathways utilized during orofacial development.

Animals↗
PubMed2004

Structure-composition relations for the partly disordered Hume-Rothery phase Ir7+7deltaZn97-11delta (0.31< or =delta< or =0.58).

The crystal structure, its variation within the homogeneity range and some physical properties of the new zinc-rich, partly disordered phase Ir7+7deltaZn97-11delta (0.31< or =delta< or =0.58) are reported. The structures of three phases with distinct composition were determined by means of single crystal X-ray diffraction. Ir7+7deltaZn97-11delta exhibits a significant homogeneity range, adopts a complex gamma-brass related cubic structure (cF403-406), is stable up to 1201(2) K, and transforms sluggishly below 1048(4) K into a phase with 394 atoms in the monoclinic primitive unit cell. It is a diamagnetic, moderate metallic conductor. Six distinguishable clusters consisting of 22-29 atoms comprise the structure. The clusters are situated about the 16 high symmetry points of the cubic F lattice. The structure can be subdivided into two partial structures, one with constant composition IrZn5 and 192 atoms per unit cell and a second being significantly richer in zinc with variable composition and 211-214 atoms per unit cell. The meandering triply periodic minimal surface of two interpenetrating diamond-like nets separates the compositionally variable from its complementary invariant part. The phase width is coupled with substitutional and positional disorder. A comprehensive analysis of composition-dependent site occupancy factors reveals a linear correlation between the various types of disorder which can be conclusively interpreted in terms of an incoherent intergrowth of distinctive partial structures in variable proportions on a length scale comparable to the size of the approximately 2 nm large unit cell. On the basis of the structural findings we derive the structure chemically meaningful formula Ir7+7delta)Zn97-11delta which quantitatively accounts for the interrelation between substitutional and positional disorder and provides a measure for the homogeneity range in structural terms.

Alloys↗
PubMed2002

Myosins of Babesia bovis: molecular characterisation, erythrocyte invasion, and phylogeny.

Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50-60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35-39%) with all members of Class XIV, and 5'-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite.

Actins↗
PubMed2006

Pathologic findings from the Breast Cancer Surveillance Consortium: population-based outcomes in women undergoing biopsy after screening mammography.

BACKGROUND: To the authors' knowledge, a comprehensive analysis of pathology outcomes after screening mammography, as it is practiced clinically in the U.S. general population, has not been performed. METHODS: Breast Cancer Surveillance Consortium data from 1996-2001 were used to identify pathology specimens that were obtained within 1 year of screening mammograms performed on 786,846 women ages 40-89 years. The pathology results were classified as invasive carcinoma, ductal carcinoma in situ (DCIS), or benign. The associations between overall pathology outcomes and invasive tumor size and lymph node status were analyzed by age and mammography assessment category. RESULTS: The rates of both recommending and performing a biopsy varied little with age. The 1,664,032 screening mammograms were followed by 26,748 known biopsies (1.6%) and 8815 diagnoses of breast carcinoma (0.53%). Overall, 81% of carcinomas were invasive, and 78% of those were pathologically lymph node-negative tumors, in contrast to the 66% prevalence observed in the Surveillance, Epidemiology, and End Results (SEER) data during the same period. Most invasive tumors measured between 0 mm and 10 mm (35%) or between 11 mm and 20 mm (36%) in greatest dimension, and 92% and 78% were lymph node-negative tumors, respectively: Biopsy results that were classified as malignant increased with age (P < 0.0001) and were most likely to follow Breast Imaging, Reporting, and Diagnosis System Category 5 and 4 assessments, respectively. Ductal hyperplasia (19.6%), fibroadenoma (18.5%), and other benign findings (56.1%) were the most common benign diagnoses. CONCLUSIONS: Pathologically negative lymph nodes were more prevalent in this mammographically screened population than in the overall SEER population. The prevalence of invasive carcinoma, DCIS, and benign findings presented herein establish a range of expected biopsy outcomes for women after screening mammography in the general U.S. population.

Adult↗
PubMed2001

In vivo expression and localization of the fibroblast growth factor system in the intact and lesioned rat peripheral nerve and spinal ganglia.

Basic fibroblast growth factor (FGF-2) is involved in several cellular processes of the nervous system during development, maintenance, and regeneration. In the central nervous system, FGF-2 has been shown to be expressed in neurons and glial cells, depending on the developmental stage and brain area. In the present study, a comprehensive analysis was performed of the cellular distribution of the transcripts of FGF-2 and of the FGF high-affinity receptors (R) 1-4 in intact and lesioned sciatic nerve and spinal ganglia. In the adult rat sciatic nerve FGF-2, FGFR1-3 were expressed at low levels as revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). Sciatic nerve crush resulted in an increase of these transcript levels. FGFR4 expression was not detected in the intact and crushed nerve as revealed by RT-PCR and RNase protection assay. In situ hybridization using riboprobes for FGF-2, FGFR1-3 displayed staining in diverse cell types. Immunocytochemical staining of consecutive sections with cell markers for myelin, macrophages, and neurons revealed colocalization of the transcripts with Schwann cells and macrophages. In addition to FGF-2 and FGFR1, the transcripts of FGFR2-4 were expressed in neurons of spinal ganglia. Crush lesion of the sciatic nerve resulted in no alterations of the FGFR1-4 transcripts, whereas FGF-2 and FGFR3 mRNAs were up-regulated in spinal ganglia. The expression of FGFRs and FGF-2 in Schwann cells and macrophages at the lesion site of the sciatic nerve and in sensory neurons suggests that FGF-2 is involved in specific functions of these cells during regeneration.

Animals↗
PubMed2007

Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study.

Retinal degeneration 10 (rd10) mice are a model of autosomal recessive retinitis pigmentosa (RP), identified by Chang et al. in 2002 (Vision Res. 42:517-525). These mice carry a spontaneous mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. The aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP. We analyzed the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also studied retinal function with the electroretinogram (ERG), recorded between P18 and P30. We found that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells. Thus, changes in the rd10 retina are very similar to what was previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predicts that rd10 mice might become excellent models for rescue approaches.

Animals↗
PubMed2007

Selective enrichment of DJ-1 protein in primate striatal neuronal processes: implications for Parkinson's disease.

Mutations in DJ-1 cause autosomal recessive, early-onset Parkinson's disease (PD). The precise function and distribution of DJ-1 in the central nervous system remain unclear. In this study, we performed a comprehensive analysis of DJ-1 expression in human, monkey, and rat brains with antibodies that recognize distinct, evolutionarily conserved epitopes of DJ-1. We found that DJ-1 displays region-specific neuronal and glial labeling in human and nonhuman primate brain, sharply contrasting with the primarily neuronal expression pattern observed throughout rat brain. Further immunohistochemical analysis of DJ-1 expression in human and nonhuman primate brains showed that DJ-1 protein is expressed in neurons within the substantia nigra pars compacta and striatum, two regions critically involved in PD pathogenesis. Moreover, immunoelectron microscopic analysis revealed a selective enrichment of DJ-1 within primate striatal axons, presynaptic terminals, and dendritic spines with respect to the DJ-1 expression in prefrontal cortex. Together, these findings indicate neuronal and synaptic expression of DJ-1 in primate subcortical brain regions and suggest a physiological role for DJ-1 in the survival and/or function of nigral-striatal neurons.

Aged↗
PubMed1995

On the importance of evolution to developmental psychobiology.

The term developmental psychobiology implies an integration between psychology and biology. But what segment of biology does the discipline embrace? The present commentary asserts that developmental psychobiology devotes too much attention to structural biology, with its emphasis on proximate mechanisms, and fails to give enough prominence to evolutionary biology and ultimate perspectives. I have attempted to portray the significance of evolution to developmental psychobiology and to elaborate on how developmental psychobiology might contribute to refinements in evolutionary theory, especially recent modifications that advocate a greater role for developmental processes. Methodological suggestions are offered, which would broaden developmental psychobiology's perspective so that a more comprehensive analysis of behavioral development results.

Aging↗
PubMed2006

Branch to colony trajectory in a modular organism: pattern formation in the Indo-Pacific coral Stylophora pistillata.

The architecture of the colony in a branching coral is an iterative process in which new layers of calcium carbonate compile atop existing structures that remain unchanged. Colony growth and development, known as astogeny, is believed to be a continuous process, characterized by replication of lower rank unites, polyps, and branches. This study seeks to explore the genetic blueprint of branch-to-colony developmental trajectory in the branching coral Stylophora pistillata, within an astogeny period of 1 year. One hundred small branches (initially 2-4 cm long) were sampled from 10 colonies. A year later, 63 remaining colonies were analyzed for their architectural rules by using 15 morphometric parameters. Multivariate statistical tests were preformed. Cluster and two-dimensional nonmetric Multi-Dimensional Scaling analyses revealed that the 10 genotypes could be divided into two major morphometric groups and two intermediate groups, whereas SIMPER analyses (a similarity percentage test) on within-genet similarities showed high similarity between the ramets developed from each of the 10 genotypes. Although, at first, it seemed that different colonies exhibited variable and different architectural designs (each characterized by specific morphometric parameters), a comprehensive analysis revealed that all 10 coral genotypes exhibited a single common developmental plan that was characterized by a continuum of architectural design with several distinct stages. Each stage is marked by its own characteristic morphometric parameters. Changing of developmental rules during the trajectory from branch to coral colony may help the colony to cope better with environmental constraints.

Animals↗
PubMed2006

Birth and death of cells in limb development: a mapping study.

Cell death and cell proliferation are basic cellular processes that need to be precisely controlled during embryonic development. The developing vertebrate limb illustrates particularly well how correct morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Precise knowledge of the patterns of cell proliferation and cell death during limb development is required to understand how their modifications may contribute to the generation of the great diversity of limb phenotypes that result from spontaneous mutations or induced genetic manipulations. We have performed a comprehensive analysis of the patterns of cell death, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL), and cell proliferation, assayed by anti-phosphorylated histone H3 immunohistochemistry, in consecutive sections of forelimbs and hindlimbs covering an extensive period of chick and mouse limb development. Our results confirm and expand previous reports and show common and specific areas of cell death for each species. Mitotic cells were found scattered in a uniform distribution across the early limb bud, with the exception of the areas of cell death in which mitotic cells were scarce. At later stages, mitotic cells were seen more abundantly in the digital tips. The aim of the present study was to satisfy the need for organized data sets describing these processes, which will allow the side-by-side comparison between the two major model organisms of limb development, i.e., the mouse and the chick.

Animals↗
PubMed1984

Characterization of human granular lymphocyte subpopulations expressing HNK-1 (Leu-7) and Leu-11 antigens in the blood and lymphoid tissues from fetuses, neonates and adults.

Three subpopulations of human granular lymphocytes from blood and lymphoid tissues were characterized using combinations of the monoclonal antibodies (mAb) HNK-1 (Leu-7), Leu-11 and VEP13. Each subpopulation was confirmed to possess natural killer (NK) cell functional capability, but a different level of cytotoxic efficiency (HNK-1+leu-11- less than HNK-1+Leu-11+ less than HNK-1-Leu-11+). In adult from 23 healthy donors, the subpopulations with HNK-1+Leu-11-, HNK-1+Leu-11+ and HNK-1-Leu-11+ phenotypes comprised 4.7 +/- 3.0, 8.0 +/- 6.4 and 3.9 +/- 3.5% of mononuclear cells, respectively. Despite their distinct surface marker phenotypes and NK functional ability, all 3 subpopulations exhibited granular lymphocyte morphology. One of these subpopulations, HNK-1+Leu-11-, also expressed the pan-T cell antigen Leu-4. Different patterns were observed in fetal bone marrow and cord blood, where the vast majority of HNK-1+ cells lacked the Leu-11 antigen (HNK-1+Leu-11+ cells). The HNK-1 antigen was not expressed on granulocytes and their precursors, whereas both Leu-11 and VEP13 antigens were expressed on these myeloid cells from fetal bone marrow and cord blood as well as adult bone marrow and spleen. Cell lines of granular lymphocytes cultured in the presence of interleukin 2 all possessed the HNK-1+Leu-4+ phenotype and NK functional capability but lacked the Leu-11 and VEP13 antigens on their surface after 15 days of culture. Although granular lymphocytes expressing the Fc receptors reacting with the mAb Leu-11 and VEP13, are the most functionally active NK cells, the HNK-1+ subpopulation lacking the Leu-11 and VEP13 antigens appears to be an important population (possibly an immature form of granular lymphocytes) for delineating the cell lineage(s) and differentiation of human granular lymphocytes. Although none of the currently available mAb react both inclusively and exclusively with human granular lymphocytes, the combination usage of these antibodies permits a more precise and comprehensive analysis of these subsets.

Adolescent↗
PubMed1992

Memory T lymphocyte hyporesponsiveness to non-cognate stimuli: a key factor in age-related immunodeficiency.

Previous studies from our laboratory have suggested that aging leads to an accumulation of cells expressing high levels of CD44, thought to be a marker for memory lymphocytes, and that positively selected CD44hi T cells, from mice of any age, respond poorly to concanavalin A (Con A) in limiting dilution estimates of interleukin (IL)-2-producing cells. We now report the results of a more comprehensive analysis of memory T cell function, in old and young mice, to non-cognate activators (Con A and the staphylococcal enterotoxin SEB). We report that memory T cells, isolated by removing cells bearing the CD45RB determinant, contain very few cells able to respond to either Con A or SEB under limiting dilution culture conditions, whether the responses are measured by IL-2 or by IL-3 accumulation. As a control, we show that memory T cells do respond strongly, at limiting dilution, to recently encountered priming antigens, i.e. Schistosoma mansoni egg antigen; the limiting dilution culture protocol thus does not preclude activation of memory T cells when cognate stimuli are presented to antigen-specific cells. These data suggest that virgin and memory T cells may differ fundamentally in their activation requirements, and suggest further that the accumulation, with age, of memory T cells accounts for the low responsiveness of old mice to non-cognate mitogens.

Aging↗
PubMed2006

Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background.

Protein expression alterations unrelated to an investigated phenotype are accumulated in most cell line models during establishment. Performing a whole proteome screening of lymphoma cell lines, we established a method to reduce the influence of protein expression unrelated to the distinct investigated phenotype. In 2-D PAGE, the comprehensive analysis of a large number of protein spots would be simplified by pooling cell line samples of the investigated phenotype. Applying this pooling approach, unrelated alterations of single samples are 'muted' by dilution. Analysing two different lymphoma subtypes (follicular and mantle cell lymphoma) by this method, spots originating in only single cell lines were reduced by 72% (650/900), whereas even modestly altered expression of protein spots detected in all lines were reliably detected in the pooled protein gels. We conclude that our pooling approach is a preferable approach to reliably detect a common protein expression pattern and may even allow proteomic analysis of clinical samples with limited amounts of sample material, even with minimal cell numbers as low as 1 x 10(6).

Cell Line, Tumor↗
PubMed2002

Identification of cytogenetic subclasses and recurring chromosomal aberrations in AML and MDS with complex karyotypes using M-FISH.

Complex chromosomal aberrations (CCAs) can be detected in a substantial proportion of AML and MDS patients, de novo as well as secondary or therapy-related, and are associated with an adverse prognosis. Comprehensive analysis of the chromosomal rearrangements in these complex karyotypes has been hampered by the limitations of conventional cytogenetics. As a result, our knowledge concerning the cytogenetics of these malignancies is sparse. Here we describe a multiplex-FISH (M-FISH) study of CCAs in 36 patients with AML and MDS. M-FISH generated a genome-wide analysis of chromosomal aberrations in CCAs, establishing several cytogenetic subgroups. -5/5q- was demonstrated in the majority of patients (86%). Other rearrangements (present with or without -5/5q-) included: deletion of 7q (47%), 3q rearrangements (19%), and MLL copy gain or amplification (17%). These genetic subgroups seem to display biological heterogeneity: MLL copy gain or amplification in association with 5q- was detected only in AML patients and was significantly associated with extremely short survival (median overall survival: 30 days, P = 0.0102). A partially cryptic t(4;5)(q31;q31), a balanced t(1;8)(p31;q22), and an unbalanced der(7)t(7;14)(q21;q13) were detected as possible new recurrent rearrangements in association with CCAs. Novel reciprocal translocations included t(5;11)(q33;p15)del(5)(q13q31) and t(3;6)(q26;q25). We conclude that AML and MDS with CCAs can be subdivided into molecular cytogenetic subclasses, which could reflect different clinical behavior and prognosis, and that three recurrent chromosomal aberrations are associated with karyotype complexity.

Acute Disease↗