Biomedical subjects

R Levy

Publications and source records attributed to R Levy.

At least 91 records · Page 5Linked to original sources

PubMed2001

Superantigen antagonist blocks Th1 cytokine gene induction and lethal shock.

Bacterial superantigens trigger an excessive, Th1-cytokine response leading to toxic shock. We designed a peptide antagonist that inhibits SEB-induced expression of human genes for IL-2, IFN-gamma, and TNF-beta, cytokines that mediate shock. The peptide antagonist shows homology to a beta-strand-hinge-alpha-helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus and Streptococcus pyogenes yet remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. For Th1-cell activation, superantigens depend on this domain. The peptide protected mice against lethal challenge with SEB or SEA. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed broad-spectrum, protective immunity, which rendered them resistant to further lethal challenges with different staphylococcal and streptococcal superantigens. Thus, the lethal effect of superantigens, mediated by Th1 cytokines, can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade, before activation of T cells takes place.

Amino Acid Motifs↗

PubMed2001

Serum and mucosal immunologic responses in children following the administration of a new inactivated intranasal anti-influenza vaccine.

Children are at considerable risk for influenza infection and may constitute the main vector for transmitting the virus to adults in the community. At present, the use of available vaccines in children is limited mainly because of a fear of side effects from the injection. Intranasal immunization was assessed as a painless, side effect-free method of facilitating the enrollment of children in vaccination programs. One intranasal dose of a trivalent inactive whole virus vaccine containing 20 microg of the three recommended seasonal viral strains was administered to 28 children recruited over two separate winter periods (1997/1998 and 1998/1999). No adverse effects were recorded. Serum IgG responses were determined by the hemagglutination inhibition (HI) method and nasal IgA responses by enzyme-linked immunosorbent assay (ELISA). In both study period seasons, 77.7%-94.4% of children were found to be immune. There was a 3.7 x and 4.7 x increase in geometric mean titer (GMT) for A/H3N2 strains, 1.9 x and 3.9 x for A/H1N1 strains, and a 3.2 x and 1.7 x for B strains in 1997/1998 and 1998/1999, respectively. The increase in GMT, as well as fourfold increases in titer level, was higher when calculated among the nonimmune children prior to vaccination. Of these, 50%-87.5% became immune following immunization. Local antibody response to the three viral strains was detected in 50%-55% of the immunized children. Also, 83.3%, 73.3%, and 61.1% of the vaccinees exhibited a mucosal and/or serum antibody response to the A/Beijing, A/Sydney, and B/Harbin strains, respectively. This mucosal response may forestall influenza development in its early stages, thereby contributing significantly to the reduction of influenza spread in the community.

Administration, Intranasal↗

PubMed2000

The inference of antigen selection on Ig genes.

Analysis of somatic mutations in V regions of Ig genes is important for understanding various biological processes. It is customary to estimate Ag selection on Ig genes by assessment of replacement (R) as opposed to silent (S) mutations in the complementary-determining regions and S as opposed to R mutations in the framework regions. In the past such an evaluation was performed using a binomial distribution model equation, which is inappropriate for Ig genes in which mutations have four different distribution possibilities (R and S mutations in the complementary-determining region and/or framework regions of the gene). In the present work, we propose a multinomial distribution model for assessment of Ag selection. Side-by-side application of multinomial and binomial models on 86 previously established Ig sequences disclosed 8 discrepancies, leading to opposite statistical conclusions about Ag selection. We suggest the use of the multinomial model for all future analysis of Ag selection.

Antigens↗

PubMed2000

In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex.

The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.

Animals↗

PubMed2000

High-frequency synchronization of neuronal activity in the subthalamic nucleus of parkinsonian patients with limb tremor.

It has been hypothesized that in Parkinson's disease (PD) there is increased synchronization of neuronal firing in the basal ganglia. This study examines the discharge activity of 121 pairs of subthalamic nucleus (STN) neurons in nine PD patients undergoing functional stereotactic mapping. Four patients had a previous pallidotomy. A double microelectrode setup was used to simultaneously record from two neurons separated by distances as small as 250 micrometer. In the six patients who had limb tremor during the recording session (n = 76 pairs), the discharge pattern of 12 pairs of tremor cells (TCs) was found to be coherent at the frequency of the limb tremor. Both in-phase and out-of-phase relationships were observed between TCs. Interestingly, in these six patients, 63/129 single neurons displayed 15-30 Hz oscillations, whereas 36/76 pairs were coherent in this frequency range. Although the oscillatory frequencies were variable between patients, they were highly clustered within a patient. The phase difference between these pairs was found to be close to 0. High-frequency synchronization was observed during periods of limb tremor as well as during intermittent periods with no apparent limb tremor. In contrast, in the three patients without limb tremor during the recording session, only 1/84 neurons had high-frequency oscillatory activity, and no TCs or synchronous high-frequency oscillatory activity was observed (n = 45 pairs). These findings demonstrate that in PD patients with limb tremor, many STN neurons display high-frequency oscillations with a high degree of in-phase synchrony. The results suggest that high-frequency synchronized oscillatory activity may be associated with the pathology that gives rise to tremor in PD patients.

Action Potentials↗

PubMed2000

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas.

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Biopsy↗

PubMed2000

'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns.

BACKGROUND: Large gene expression studies, such as those conducted using DNA arrays, often provide millions of different pieces of data. To address the problem of analyzing such data, we describe a statistical method, which we have called 'gene shaving'. The method identifies subsets of genes with coherent expression patterns and large variation across conditions. Gene shaving differs from hierarchical clustering and other widely used methods for analyzing gene expression studies in that genes may belong to more than one cluster, and the clustering may be supervised by an outcome measure. The technique can be 'unsupervised', that is, the genes and samples are treated as unlabeled, or partially or fully supervised by using known properties of the genes or samples to assist in finding meaningful groupings. RESULTS: We illustrate the use of the gene shaving method to analyze gene expression measurements made on samples from patients with diffuse large B-cell lymphoma. The method identifies a small cluster of genes whose expression is highly predictive of survival. CONCLUSIONS: The gene shaving method is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worth further investigation.

Algorithms↗

PubMed2000

Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5' noncoding regulatory region of the BCL-6 gene.

Follicle center lymphoma (FCL) is an indolent low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms to aggressive diffuse large B-cell lymphoma (DLBCL). Histologic transformation of FCL is commonly associated with accumulation of secondary genetic alterations. The BCL-6 gene is altered by chromosomal rearrangements and mutations clustering in its 5' noncoding regulatory region in up to 70% of primary DLBCL, but in a significantly smaller subset of FCL. Previous studies have shown that both chromosomal rearrangements and mutations could deregulate BCL-6 expression. To evaluate the association between progressive accumulation of BCL-6 regulatory region mutations and the histologic transformation of FCL, we analyzed by extensive cloning and sequencing paired biopsy specimens obtained at the time of FCL diagnosis and transformation (6 patients) or FCL relapse (3 patients). In an additional patient, biopsy specimens obtained at the time of diagnosis, FCL relapse, and subsequent transformation to DLBCL were evaluated. The presence of identical mutations in the paired diagnosis and posttransformation DLBCL specimens confirmed the common clonal origin of both the pretransformation and the posttransformation lymphomas. No new mutations in the 5' noncoding regulatory region of the BCL-6 gene were detected in any of the specimens evaluated at the time of FCL relapse. In contrast, 5 of the 7 transformed specimens contained new mutations not found in the paired original biopsy specimens obtained at the time of FCL diagnosis or relapse. The number of these new mutations ranged from 1 to 6 per specimen. Some of the new mutations tended to cluster in certain areas of the 5' noncoding regulatory region of the BCL-6 gene. Our results show that transformation of FCL to DLBCL is associated with accumulation of new mutations in the 5' noncoding regulatory region of the BCL-6 gene, that by deregulation of the BCL-6 gene expression may play a role in lymphoma transformation. (Blood. 2000;96:635-639)

Biopsy↗

PubMed2000

Recombinant adenovirus vaccine encoding a chimeric T-cell antigen receptor induces protective immunity against a T-cell lymphoma.

Vaccination using recombinant tumor-derived T-cell antigen receptor (TCR) protein induces a protective, idiotype-specific immune response against a murine T-cell tumor. However, the technically demanding task of producing patient-specific, recombinant TCR protein restricts the translation of TCR vaccines for clinical use. We report here the development of an effective recombinant TCR adenovirus vaccine. Individual adenoviruses were constructed to encode a chimeric TCR derived from either tumor Valpha or Vbeta regions fused to xenogeneic human constant regions. Coinjection of the chimeric alpha- and the beta-TCR adenoviruses protected mice against tumors. The level of protection was comparable to that achieved by an optimized regimen of recombinant TCR protein vaccines. Tumor immunity induced by TCR adenoviruses required the xenogeneic constant regions and was mediated by CD8+ T cells. Independent vaccines consisting of adenovirus expressing either chimeric alpha- or beta-TCR chain also stimulated a protective immune response. Immunization with TCR adenovirus may offer a new efficacious, protein-free vaccination approach for the treatment of T-cell malignancies.

Animals↗

PubMed2000

Linkage of foreign carrier protein to a self-tumor antigen enhances the immunogenicity of a pulsed dendritic cell vaccine.

The unique Ag-presenting capabilities of dendritic cells (DCs) make them attractive vehicles for the delivery of therapeutic cancer vaccines. While tumor Ag-pulsed DC vaccination has shown promising results in a variety of murine tumor models and early clinical trials, the optimal form of tumor Ag for use in DC pulsing has not been determined. We have studied DC vaccination using alternative forms of a soluble protein tumor Ag, the tumor-specific Ig idiotype (Id) expressed by a murine B cell lymphoma. Vaccination of mice with Id-pulsed DCs was able to induce anti-Id Abs only when the Id was modified to constitute a hapten-carrier system. DCs pulsed with Id proteins modified to include foreign constant regions, foreign constant regions plus GM-CSF, or linkage to keyhole limpet hemocyanin (KLH) carrier protein were increasingly potent in their ability to elicit anti-Id Abs. Vaccination with Id-KLH-pulsed DCs induced tumor-protective immunity superior to that obtained with Id-KLH plus a chemical adjuvant, and protection was not dependent upon effector T cells. Rather, protection was associated with the induction of high titers of anti-Id Abs of the IgG2a subclass, characteristic of a Th1 response. These findings have implications for the design of therapeutic Ag-pulsed DC vaccines for cancer immunotherapy in humans.

Adjuvants, Immunologic↗

PubMed2000

The requirement of both extracellular regulated kinase and p38 mitogen-activated protein kinase for stimulation of cytosolic phospholipase A(2) activity by either FcgammaRIIA or FcgammaRIIIB in human neutrophils. A possible role for Pyk2 but not for the Grb2-Sos-Shc complex.

The signal transduction pathways initiated by opsonized zymosan (OZ) leading to activation of cytosolic phospholipase A(2) (cPLA(2)) in human neutrophils remain obscure. In a previous study, we showed that the activation of cPLA(2) by OZ is tyrosine kinase-dependent. The present study demonstrates that the signals initiated by OZ involve activation of tyrosine kinase Pyk2 but not the formation of the adhesion protein complex, Shc-Grb2-Sos. Stimulation of cPLA(2) activity by OZ is mediated by Fc gamma receptors (FcgammaRs) and not by complement receptors for the C3b protein. Cross-linking of FcgammaRIIA or FcgammaRIIIB induces p38 mitogen-activated protein (MAP) kinase and extracellular regulated kinase (ERK) phosphorylation. The kinetics of cPLA(2) activity stimulated by either of the FcgammaRs or by both is similar to that of p38 MAP kinase and was detected as early as 15 s after stimulation, maintained a plateau for 10 min, and decreased thereafter. ERK activation was detected also within 15 s but decreased significantly 5 min after stimulation. The MEK inhibitor, PD-098059, or the p38 MAP kinase inhibitor, SB-203580, caused a partial inhibition during the time course of cPLA(2) activity, whereas their combination caused a total inhibition. Thus, although ERK activation is significantly shorter than that of p38 MAP kinase, it is equally required for activation and maintenance of cPLA(2) activity by occupancy of a single receptor, FcgammaRIIA or FcgammaRIIIB.

Antigens, CD↗

PubMed2000

NMR structure of an anti-gp120 antibody complex with a V3 peptide reveals a surface important for co-receptor binding.

BACKGROUND: The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS: The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

Amino Acid Sequence↗

PubMed2000

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration.

Morphine-3- and morphine-6-glucuronide are morphine's major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Molecular analysis of immunoglobulin genes in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is a common type of non-Hodgkin's lymphoma (NHL) that is highly heterogeneous from both clinical and histopathologic viewpoints. The immunoglobulin (Ig) heavy (H) chain variable region genes were examined in 71 patients with untreated primary DLBCL. Fifty-eight potentially functional V(H) genes were detected in 53 DLBCL cases; V(H) genes were nonfunctional in 9 cases and were not detected in an additional 9 cases. The use of V(H) gene families by DLBCL tumors was unbiased without overrepresentation of any particular V(H) gene or gene family. Analysis of Ig mutations in comparison to the most closely related germline gene disclosed mutated V(H) genes in all but 1 DLBCL case. More than 2% difference from the most similar germline sequence was detected in 52 potentially functional and the 8 nonfunctional V(H) gene sequences, whereas less than 2% difference from the germline sequence was observed in 3 V(H) gene isolates. Only 3 V(H) gene isolates were unmutated. No correlation was found between V(H) gene use, mutation level, and International Prognostic Index (IPI) or survival. Six of 8 tested tumors showed evidence of ongoing somatic mutations. Evidence for positive or negative antigen selection pressure was observed in 65% of mutated DLBCL cases. Our findings indicate that the etiology and the driving forces for clonal expansion are heterogeneous, which may explain the well-known clinical and pathologic heterogeneity of DLBCL. (Blood. 2000;95:1797-1803)

B-Lymphocytes↗

PubMed2000

Mutation analysis of the 5' noncoding regulatory region of the BCL-6 gene in non-Hodgkin lymphoma: evidence for recurrent mutations and intraclonal heterogeneity.

The BCL-6 proto-oncogene is involved in the genesis of non-Hodgkin lymphoma (NHL). Rearrangements due to chromosomal translocations and somatic mutations of the 5' noncoding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. To further elucidate the nature of the somatic mutations in the regulatory region of this gene, we have studied 10 healthy donors and 11 NHL biopsy samples by extensive molecular cloning and sequencing. In addition, we analyzed the BCL-6 genes of tumor and nontumor cells from 2 of the cases. The germ line sequence of this region was defined, which differs in 7 positions from that previously reported. In addition, 1 polymorphic variation at position 397(G or C) was identified. Deletions, insertions, and repeated substitution mutations were detected among the molecular isolates in 8 tumor specimens, with a mutational incidence ranging from 1.3 x 10(-3) to 1.3 x 10(-2)/bp (base pair). A total of 20 distinct substitution mutations, 1 insertion and 3 deletions were observed. One of these deletion mutations and 2 of the substitutions were observed in more than 1 tumor specimen from different individuals. In 3 tumor samples, identical mutations affecting both alleles were observed. These findings suggest the presence of mutational hot spots and hot specific events, a finding supported by our compilation of previously published data. In 6 samples, the nucleotide sequences showed evidence of intraclonal heterogeneity, consistent with a stepwise ongoing mutational process affecting the BCL-6 gene in the tumor cells. These mutations accumulating in the regulatory region of the BCL-6 gene could play a role in lymphoma progression and in the transformation of follicular lymphomas to more aggressive large cell lymphomas. (Blood. 2000;95:1400-1405)

Base Sequence↗

PubMed2000

[Right heart failure caused by thiamine deficiency (cardiac beriberi)].

BACKGROUND: Vitamin B1 deficiency (beriberi) is very uncommon in France. It leads to high output cardiac failure totally different from the situation observed in alcoholic patients. We report a typical case of cardiac beriberi. CASE REPORT: The patient was referred for dyspnea with high output cardiac failure. Echocardiography evidenced severe pulmonary hypertension and high cardiac output. The more common causes of heart failure were ruled out. The dietary habits of the patient (suggested beriberi which was confirmed by the low serum thiamin and therapeutic test with vitamin B1. DISCUSSION: High output cardiac failure should suggest possible Shoshin beriberi, particularly in subjects with imported dietary habits living in a precarious socioeconomic situation.

Beriberi↗

PubMed2000

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Adult↗

PubMed2000

Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor.

The DRY motif, at the junction of transmembrane helix 3 and intracellular loop 2 of G protein-coupled receptors, is highly conserved. Mutations were introduced into the CB2 cannabinoid receptor to study the role of this motif in CB2 signaling. D mutations (DRY130-132AAA and D130A) markedly reduced binding of cannabinoid agonists, while no significant reduction was observed with R131A or Y132A. Mutating R (R131A) only partially reduced, and mutating Y (Y132A) more efficiently reduced the cannabinoid-induced inhibition of adenylyl cyclase. Thus, in CB2, D130 is involved in agonist binding, whereas Y seems to have a role in receptor downstream signaling.

Amino Acid Motifs↗