PubMed Health⌕ Search

Biomedical subjects

Peter E Lipsky

Publications and source records attributed to Peter E Lipsky.

At least 55 records · Page 3Linked to original sources
PubMed2004

Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER.

We analyzed 77 nonproductive and 574 productive human V(H)DJ(H) rearrangements with a newly developed program, JOINSOLVER. In the productive repertoire, the H chain complementarity determining region 3 (CDR3(H)) was significantly shorter (46.7 +/- 0.5 nucleotides) than in the nonproductive repertoire (53.8 +/- 1.9 nucleotides) because of the tendency to select rearrangements with less TdT activity and shorter D segments. Using criteria established by Monte Carlo simulations, D segments could be identified in 71.4% of nonproductive and 64.4% of productive rearrangements, with a mean of 17.6 +/- 0.7 and 14.6 +/- 0.2 retained germline nucleotides, respectively. Eight of 27 D segments were used more frequently than expected in the nonproductive repertoire, whereas 3 D segments were positively selected and 3 were negatively selected, indicating that both molecular mechanisms and selection biased the D segment usage. There was no bias for D segment reading frame (RF) use in the nonproductive repertoire, whereas negative selection of the RFs encoding stop codons and positive selection of RF2 that frequently encodes hydrophilic amino acids were noted in the productive repertoire. Except for serine, there was no consistent selection or expression of hydrophilic amino acids. A bias toward the pairing of 5' D segments with 3' J(H) segments was observed in the nonproductive but not the productive repertoire, whereas V(H) usage was random. Rearrangements using inverted D segments, DIR family segments, chromosome 15 D segments and multiple D segments were found infrequently. Analysis of the human CDR3(H) with JOINSOLVER has provided comprehensive information on the influences that shape this important Ag binding region of V(H) chains.

Algorithms↗
PubMed2004

Generation and regulation of human Th1-biased immune responses in vivo: a critical role for IL-4 and IL-10.

Tissue damage in many human autoimmune diseases is mediated by activated autoantigen-specific Th1 cells. Delineation of the regulatory mechanisms controlling a Th1-biased human immune reaction and its pathologic potential is, therefore, a critical step in the understanding of autoimmune diseases. In this study, we introduce a novel means to investigate human Th1-biased immune responses in vivo. Intraperitoneal injection of human mononuclear cells into immunodeficient mice generates a xenogeneic Th1-biased human immune response characterized by systemic inflammation and leukocytic infiltrates with a granuloma-like architecture in the liver, and the perigastrointestinal and perirenal fatty tissue. Th1 cell activation was dependent on the presence of APCs and could be blocked by cyclosporine. Importantly, neutralization of endogenously produced IL-4 and IL-10 markedly exaggerated the immune response, whereas exogenous IL-4 and IL-10 inhibited systemic Th1 immunity. Thus, the model described in this paper presents a useful means to analyze the regulation of human immune reactions in an in vivo situation. The results suggest that both IL-4 and IL-10 contribute to controlling the development of a human Th1-biased immune reaction.

Animals↗
PubMed2004

Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals.

Abnormalities in lymphocyte signaling cascades are thought to play an important role in the development of autoimmune disease. However, the large amount of cellular material needed for standard biochemical assessment of signaling status has made it difficult to evaluate putative abnormalities completely using primary lymphocytes. The development of technology to employ intracellular staining and flow cytometry to assess the signaling status of individual cells has now made it possible to delineate the perturbations that are present in lymphocytes from patients with autoimmune disease. As an example, human B cells from the Ramos B cell line and the periphery of systemic lupus erythematosus (SLE) patients or normal nonautoimmune controls were assessed for activation of the NF-kappaB and mitogen activated protein kinase (MAPK) signaling cascades by intracellular multiparameter flow cytometric analysis and biochemical Western blotting. In combination with fluorochrome conjugated antibodies specific for surface proteins that define B cell subsets, antibodies that recognize activated, or phosphorylated inhibitors of kappaB (IkappaB) as well as the extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) or p38 MAPKs were used to stain fixed and permeabilized human B cells and analyze them flow cytometrically. Examination of the known signaling pathways following engagement of CD40 on human B cells confirmed that intracellular flow cytometry and Western blotting equivalently assay CD154-induced phosphorylation and degradation of IkappaB proteins as well as phosphorylation of the MAPKs ERK, JNK and p38. In addition, B cells from the periphery of SLE patients had a more activated status immediately ex vivo as assessed by intracellular flow cytometric analysis of phosphorylated ERK, JNK and p38 when compared with B cells from the periphery of normal, nonautoimmune individuals. Together, these results indicate that multiparameter intracellular flow cytometric analysis of signaling pathways, such as the NF-kappaB and MAPK cascades, can be used routinely to assess the activation status of a small number of cells and thus delineate abnormalities in signaling molecules expressed in primary lymphocytes from patients with autoimmune disease.

Journal Article↗
PubMed2004

GATA-3 in human T cell helper type 2 development.

The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell-mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/- individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell-mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo.

Adolescent↗
PubMed2004

Abnormalities in peripheral B cell memory of patients with primary Sjögren's syndrome.

OBJECTIVE: To delineate disturbances in peripheral B cell memory in primary Sjögren's syndrome (SS). METHODS: Isotype-specific immunoglobulin (Ig) heavy-chain transcripts were analyzed in single-sorted CD19+,CD27- naive and CD19+,CD27+ memory B cells from patients with primary SS and normal healthy control subjects. RESULTS: A significantly higher frequency of B cells expressing mu-, alpha-, and/or gamma-chain transcripts were found in patients with primary SS compared with controls (58.0% versus 14.3%; P < 0.0001). Notably, 30.5% of individual B cells (for primary SS, 38.7%; for controls, 12.7% [P < 0.0001]) simultaneously expressed transcripts for different Ig heavy-chain isotypes using identical V(H)-D-J(H) rearrangements. However, these cells lacked surface expression of more than one of the respective Ig heavy-chain isotypes as well as messenger RNA (mRNA) transcripts for 2 germinal center markers, activation-induced cytidine deaminase, and Bcl-6. In contrast with the findings in normal healthy controls, peripheral B cell memory in patients with primary SS was characterized by 1) circulating CD27+ B cells expressing heavily mutated Ig V(H) transcripts (mutational frequency 8.6% versus 4.3%; P < 0.0001), 2) significantly enhanced mutational frequencies of C mu transcripts (9.6% versus 2.5%; P < 0.0001), 3) a high proportion (61.2%) of CD27+ B cells expressing transcripts for multiple Ig heavy-chain isotypes, and 4) a CD27- memory-type B cell subpopulation expressing mutated C mu transcripts. CONCLUSION: Altogether, both B cell hyperactivity and striking abnormalities in peripheral B cell memory are indicated at the single-cell mRNA level in patients with primary SS. Detection of multiple Ig heavy-chain transcripts in peripheral CD19+,CD27+ memory B cells of patients with SS may represent the abnormal retention of pre-switch mRNA transcripts in circulating post-switch B cells.

Adult↗
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↗
PubMed2004

Sensitivity of femoral orientation estimates to condylar surface and MR image plane location.

PURPOSE: To define the femoral anatomic region that provides the most reliable reference for measuring femoral orientation, from which patellofemoral and tibiofemoral orientation can be measured. MATERIALS AND METHODS: After a three-dimensional image-based osteo-alignment procedure, two independent estimates of distal femoral orientation, the anterior (AFA) and posterior femoral angles (PFA), were acquired. These sets were comprised of 31 axial femoral orientation estimates each (obtained across a region 10 mm above to 20 mm below the patellar center) from 42 normal axial magnetic resonance (MR) image sets. An analysis of variance was conducted to determine the influence of image plane location, sex, and side on femoral orientation measures. RESULTS: The AFA presented greater variability across subjects (average SD = 3.13 degrees) as compared to the PFA (average SD = 1.78 degrees ). There were significant differences in the AFA based on image plane location and sex. In contrast, no differences were found between sides, sexes, or image plane locations for the PFA. CONCLUSION: In total, the posterior femoral condylar surface is more reliable than the anterior condylar surface for measuring femoral orientation and is best measured at least 8 mm below the patellar center.

Adult↗
PubMed2004

Biomarkers in systemic lupus erythematosus.

Despite the longstanding interest and large number of publications on biomarkers in lupus, there are no validated and widely accepted biomarkers of systemic lupus erythematosus to date. To achieve the ultimate goal, to have a biomarker as a surrogate endpoint in clinical studies, candidate biomarkers have to first be validated in a statistically rigorous way. However, to qualify as a surrogate endpoint, even validated biomarkers have to go through a process that demonstrates that they accurately reflect a clinically important outcome. These goals can only be achieved in large multicenter, properly conducted studies. We reviewed the difficulties involved in developing validated biomarkers for systemic lupus erythematosus and summarized the available data on the most promising biomarker candidates of disease susceptibility and disease activity. We also report on the current status of a multicenter initiative to concentrate efforts of biomarker development.

Biomarkers↗
PubMed2004

Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry.

A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6.

Apoptosis↗
PubMed2004

Specific in vivo deletion of B-cell subpopulations expressing human immunoglobulins by the B-cell superantigen protein L.

Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.

Animals↗
PubMed2003

A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib.

Mutational analysis of the c-kit gene in a patient with a previously undescribed variant of mast cell disease revealed a germline mutation, Phe522Cys, within the transmembrane portion of the Kit receptor protein. Transfection experiments revealed that the mutation caused ligand-independent autophosphorylation of Kit, which was inhibited by the tyrosine kinase inhibitor imatinib mesylate. The patient's bone marrow biopsy and aspirate displayed unique pathologic features with the presence of excessive numbers of mature-appearing mast cells and absence of aberrant mast cell surface expression of CD2, CD25, and CD35. Therapy with imatinib mesylate resulted in a dramatic improvement in mast cell burden and clinical symptoms. These results highlight the significance of the transmembrane region of Kit in activation of the molecule and its importance in mast cell development and suggest a role for screening for transmembrane c-kit mutations in patients with mastocytosis in association with the decision to use imatinib mesylate.

Adult↗
PubMed2003

B cell abnormalities in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.

B-Cell Activating Factor↗
PubMed2003

Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: Disease activity in systemic lupus erythematosus (SLE) is usually assessed with complex disease activity scores comprising a variety of different parameters. In order to determine whether SLE disease activity correlates with abnormal B lymphocyte activity, B cell subsets were analyzed, and their relationship to clinical and humoral measures of disease activity was assessed. METHODS: The distribution of B cell subsets was determined by fluorescence-activated cell sorting analysis and assessed in relation to the autoantibody profile, disease activity measured by the SLE Disease Activity Index (SLEDAI) and the European Consensus Lupus Activity Measure scores, disease duration, and therapy. RESULTS: The number and frequency of CD27(high) plasma cells were significantly correlated with the SLE disease activity indices and with the titer of anti-double-stranded DNA (anti-dsDNA) autoantibodies. Circulating B cell subsets were not influenced by age or sex, but appeared to relate to the duration of disease and the therapeutic regimen, with the number and frequency of CD27(high) plasma cells increasing and those of CD27- naive B cells decreasing over time. Patients were divided into those with a SLEDAI score of 0-8 (low disease activity) and those with SLEDAI score >8 (high disease activity). Patients with high disease activity had an increased frequency of both CD19+ B cells and CD27(high) plasma cells. By using a nonparametric data sieving algorithm, we observed that these B cell abnormalities provided predictive values for nonactive and active disease of 78.0% and 78.9%, respectively. The predictive value of the B cell abnormalities (78.9%) was greater than that of the humoral/clinical data pattern (71.4%), including anti-dsDNA antibody levels, circulating immune complexes, increased erythrocyte sedimentation rate, mucocutaneous involvement, and acute renal involvement. CONCLUSION: Flow cytometric monitoring of B cell subsets in the peripheral blood provides new insights into abnormalities of B cell function in SLE and may also be a diagnostically valuable option for monitoring the activity of this autoimmune disease.

Adult↗
PubMed2003

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.

BACKGROUND: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser. METHODS: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry. RESULTS: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry. CONCLUSION: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique.

Argon↗
PubMed2003

A flow cytometric method to detect protein-protein interaction in living cells by directly visualizing donor fluorophore quenching during CFP-->YFP fluorescence resonance energy transfer (FRET).

BACKGROUND: Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency. METHODS: A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by comparing CFP intensity between FRET-positive and -negative populations. To accomplish this, we compared the CFP intensity in FRET-positive and FRET-negative cells within the same population transfected with a CFP/YFP fusion protein, in which the molar ratio of CFP:YFP was one. By using separate lasers to excite CFP and YFP, the detection of FRET was separated from that of YFP. Therefore, after direct excitation, the YFP emission spectrum remained constant in all transfected cells, whereas the emission spectrum of CFP varied with the extent of FRET in individual cells. Specific CFP/YFP fusion constructs were prepared to evaluate this approach. The first one consisted of CFP and YFP separated by two caspase cleavage sites (CFP-LEVD-YFP). A second construct consisted of CFP and YFP separated by a structurally restricted 232-amino acid (aa) spacer. No FRET was observed by transfectants expressing this construct. RESULTS: Transfection of CFP-LEVD-YFP into Hela cells resulted in a FRET-positive population and a FRET-negative one. The appearance of the FRET-negative population was inhibited by the caspase inhibitor z-VAD. Moreover, substituting D for A in the caspase cleavage sites of this probe abolished the FRET-negative population, demonstrating the probe's specificity for caspase activity. Comparison of the CFP emission in the FRET-positive and FRET-negative population was used to document the relationship of FRET to donor quenching and permit the calculation of FRET efficiency and relative molecular distance between CFP and YFP. Similar results were noted when cells transfected with the caspase-sensitive probe (in the presence of z-VAD) were mixed with cells expressing the CFP-YFP construct with the 232-aa spacer and therefore were FRET negative. This demonstrated the validity of calculating CFP donor quenching and FRET efficiency by comparing emission spectra of an unknown construct with that of a known positive control, both expressed by the same population of cells. Using this approach, we confirmed that members of the TNF receptor-associated factor (TRAF) family engaged in both homotypic and heterotypic interactions. CONCLUSIONS: We have established a novel flow cytometric approach to assess donor CFP quenching during CFP-->YFP FRET, which can be used for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties. This method can be used not only to analyze cells that express a CFP and YFP fusion protein, but also independent CFP-coupled and YFP-coupled interacting proteins.

Apoptosis↗