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E Kraig

Publications and source records attributed to E Kraig.

At least 19 recordsLinked to original sources
PubMed2000

TCR v(beta) repertoire restriction and lack of CDR3 conservation implicate TCR-superantigen interactions in promoting the clonal evolution of murine thymic lymphomas.

Thymic lymphoma development is a multistage process in which genetic and epigenetic events cooperate in the emergence of a malignant clone. The notion that signaling via TCR-ligand interactions plays a role in promoting the expansion of developing neoplastic clones is a matter of debate. To investigate this issue, we determined the TCR V(beta) repertoire of thymic lymphomas induced in AKR/J mice by either endogenous retroviruses or the carcinogen, N-methyl-N-nitrosourea (MNU). Both spontaneous and MNU-induced lymphomas displayed restricted V(beta) repertoires. However, whereas V(beta)6, V(beta)8 and V(beta)9 were expressed by a greater than expected frequency of MNU-induced lymphomas, V(beta)8, V(beta)7, V(beta)13 and V(beta)14 were over-represented on spontaneous lymphomas. The dissimilar TCR V(beta) profiles indicate that different endogenous ligands promote neoplastic clonal expansion in untreated and MNU-treated mice. Although the nature of these ligands is not clear, the lack of conservation in TCR beta chain CDR3 regions among lymphomas that express the same V(beta) segment suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, are likely to be involved in the selection process. The biased representation of lymphomas expressing V(beta)6-, V(beta)7- and V(beta)9-containing TCRs that recognize endogenous SAG is consistent with this hypothesis. The finding that Bcl-2 is expressed at high levels in spontaneous and MNU-induced lymphomas suggests that preneoplastic thymocytes may be resistant to SAG-induced clonal deletion. A working model is presented in which preneoplastic clones expressing TCRs that recognize endogenous SAG are selectively expanded as a consequence of sustained TCR-mediated signaling.

Animals↗
PubMed2000

Use of defined mutants to assess the role of the Campylobacter rectus S-layer in bacterium-epithelial cell interactions.

Campylobacter rectus is a periodontal pathogen with a 150-kDa protein on its cell surface. This protein forms a paracrystalline lattice, called the S-layer, surrounding the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the possible role of the S-layer in the initial interaction of C. rectus with host epithelial cells, C. rectus strains lacking the S-layer protein gene (crsA) were constructed by allelic exchange mutagenesis. Surprisingly, the lack of the S-layer had only a minor effect on the interaction of C. rectus with HEp-2 epithelial cells; CrsA(+) cells were 30 to 50% more adherent than were CrsA(-) bacteria. Since the host cell expression of cytokines appears to play an important role in the pathogenesis of periodontal diseases, the effect of the S-layer on the epithelial cell cytokine response was also examined by quantitative reverse transcriptase PCR and enzyme-linked immunosorbent assay. Although there were no changes in the mRNA levels for the anti-inflammatory cytokines interleukin-1 receptor agonist (IL-1ra), IL-13, and transforming growth factor beta, the expression and secretion of the proinflammatory cytokines IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) were significantly induced by both wild-type C. rectus and CrsA(-) bacteria. Interestingly, the kinetics of cytokine induction differed for the CrsA(+) and CrsA(-) bacteria. At early time points, the HEp-2 cells challenged with CrsA(-) bacteria produced higher levels of IL-6, IL-8, and TNF-alpha mRNA and protein than did cells challenged with CrsA(+) bacteria. We conclude that C. rectus may help initiate periodontitis by increasing the expression of proinflammatory cytokines and that the S-layer may temper this response to facilitate the survival of C. rectus at the site of infection.

Bacterial Adhesion↗
PubMed1999

Myasthenia gravis and its animal model: T cell receptor expression in an antibody mediated autoimmune disease.

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease. Since the primary target antigen of the autoimmune response is known and a well-characterized animal model is available, MG is often considered an excellent situation for the application of novel specific immunotherapies, many of which are directed at T lymphocytes. CD4+ helper T cells are required for the development of the animal model, experimental autoimmune MG (EAMG). Even though the target antigen, acetylcholine receptor (AChR) is immunologically complex, the T cell response to AChR in mice is dominated by recognition of a single peptide by about 50% of the T cells. These T cells, in turn, utilize a restricted set of TCR gene elements and conserved CDR3 regions. While specific therapy directed at the immunodominant T cells is capable of reducing the magnitude of the anti-AChR response, considerable flexibility is apparent and reveals the ability of additional T cells to provide the requisite B cell help. In human MG patients, AChR-specific T cells have been identified but in many studies the frequencies were surprisingly low. In a very few cases, AChR-specific T cells have been cloned from MG patients. Analysis reveals heterogeneity in epitope recognition and MHC restriction. Little information on TCR structure is available. Our own studies using antigen-specific as well as non-specific methods for examining clonal T cell expansions in MG have led to an alternative hypothesis concerning T-B collaboration in MG.

Animals↗
PubMed1998

A new member of the S-layer protein family: characterization of the crs gene from Campylobacter rectus.

Strains of the periodontal pathogen Campylobacter rectus express a 150- to 166-kDa protein on their cell surface. This protein forms a paracrystalline lattice, called the surface layer (S-layer), on the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the function of the S-layer in the pathogenesis of C. rectus, we have cloned and characterized its gene. The S-layer gene (crs) from C. rectus 314 encodes a cell surface protein which does not have a cleaved signal peptide at its amino terminus. Although the amino acid sequence deduced from the crs gene has 50% identity with the amino-terminal 30 amino acids of the four S-layer proteins from Campylobacter fetus, the similarity decreases to less than 16% over the rest of the protein. Thus, the crs gene from C. rectus encodes a novel S-layer protein whose precise role in pathogenesis may differ from that of S-layer proteins from other organisms. Southern and Northern blot analyses with probes from different segments of the crs gene indicate that the S-layer gene is a single-copy, monocistronic gene in C. rectus. RNA end mapping and sequence analyses were used to define the crs promoter; there is an exact match to the Escherichia coli -10 promoter consensus sequence but only a weak match to the -35 consensus element. Southern blots of DNA from another strain of C. rectus, ATCC 33238, demonstrated that the crs gene is also present in that strain but that there are numerous restriction fragment length polymorphisms in the second half of the gene. This finding suggests that the carboxy halves of the S-layer proteins from strains 314 and 33238 differ. It remains to be determined whether the diversities in sequence are reflected in functional or antigenic differences important for the pathogenesis of different C. rectus isolates.

Amino Acid Sequence↗
PubMed1998

T cell receptor repertoire diversity and clonal expansion in human neonates.

Newborn human infants, particularly those born prematurely, are susceptible to infection with a variety of microorganisms. We questioned whether limitations in the T cell repertoire contribute to the neonatal immunocompromised state. To describe developmental changes of the T cell repertoire, cDNA segments corresponding to third complementarity regions (CDR3) of human umbilical cord blood T cell receptors (TCR) from 24-41-wk gestational age were amplified with TCR family-specific probes. The resulting amplified CDRs were visualized by fingerprinting and single strand conformation polymorphism (SSCP) analysis. At 24-wk gestation there were no limitations in TCRBV family usage, and the degree of CDR3 size heterogeneity was not different from the adult. However, earlier in gestation, CDR3s were shorter for all families and gradually increased in size until term. The extent of oligoclonal expansion observed in cord blood was greater than in adult peripheral blood (p = 0.03). T cell oligoclonal expansion was greatest at 29-33-wk gestation and declined toward term. Expansions were detectable in both CD4+ and CD8+ subpopulations. Our findings indicate that the genetic mechanisms of repertoire diversification appear intact as early as 24 wk of gestation, but repertoire diversity is limited as a result of smaller CDR3 sizes. In addition, there was a developmentally regulated progression of oligoclonally expanded T cells. These differences in the TCRBV repertoire add to the body of evidence demonstrating immaturity of the neonatal immune system. However, the role that these subtle differences are likely to play in the relative immunodeficiency of the neonate remains to be determined.

Adult↗
PubMed1997

Identification of a novel, spliced variant of CREB that is preferentially expressed in the thymus.

A conserved decamer sequence, AGTGA(T/C)(G/A)TCA, related to the cAMP response element (CRE), is found in the promoter regions of most TCR V beta gene segments. It has been shown to be a critical element for T cell-specific transcriptional regulation of the TCR genes. However, none of the decamer or CRE-binding proteins identified thus far is restricted in its expression to cells of the T cell lineage. Therefore, to find T cell-restricted decamer-binding proteins, we screened thymus cDNA expression libraries using the decamer probe. One of the thymus cDNAs identified, clone pmLY2, a novel spliced variant of CREB, was shown to be preferentially expressed in thymus. Therefore, clone pmLY2 is the first T cell-enriched decamer-binding sequence identified. We hypothesize that this CREB variant may play a role in the developmental regulation of TCR and of other T cell specific genes.

Activating Transcription Factor 2↗
PubMed1996

Evidence against chronic antigen-specific T lymphocyte activation in myasthenia gravis.

Myasthenia gravis (MG) is an antigen-specific autoimmune disease caused by antibodies against acetylcholine receptors (AChR) at the post-synaptic membrane of the neuromuscular junction. Clinical and immunological data imply the involvement of AChR-specific T lymphocytes as helper cells for autoantibody production. Direct data to support this hypothesis, however, remain sparse. In the present study, a large population of MG patients was studied for evidence of peripheral blood T cell activation by several assays. Assays based on non-specific measurements of T cell activation as well as assays of antigen-specific clonal expansion were utilized. Levels of soluble IL-2 receptor in serum were modestly elevated in some patients, suggesting T cell activation. However, peripheral blood cells did not show evidence of IL-2 receptor expression or enhanced reactivity to IL-2 in culture. Clonable T cells selected for hypoxanthine phosphoribosyl transferase (hprt) mutation, another non-antigen-specific marker for T cell activation, were not seen with increased frequency except in patients treated with purine analogs. Antigen-specific T cell activation was measured by proliferation assays using heterologous and autologous sources of AChR. Antigen-restimulated peripheral blood cell cultures were cloned by limiting dilution. The vast majority of patients failed to show convincing evidence of AChR specific T cell activation or clonal expansion; only 2 of 44 patients demonstrated clonable autologous AChR-specific T cells. An alternative hypothesis of T cell involvement in MG is proposed in which T cell activation is discontinuous and predominantly directed at antigens other than AChR.

Adult↗
PubMed1996

Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis.

Immunization of C57BL/6 mice with AChR provokes symptoms similar to those seen in the disease myasthenia gravis. To elucidate the structural requirements for T cell recognition of AChR and to identify TcR features which might provide targets for immunotherapy, a panel of T cell hybridomas was generated after immunization of mice with the immunodominant peptide of the AChR alpha chain. The TcR genes expressed by these hybridomas were sequenced. TcR-V beta 6 was preferentially employed, but other V beta genes were also observed. A conserved acidic residue was present in all CDR3 regions, regardless of the V beta. The TcR-V alpha repertoire was somewhat skewed with three V alpha families accounting for 82% of the sequences. The utilization of multiple T cell receptor V beta genes may contribute to the inability to inhibit EAMG by elimination of V beta 6+ T cells.

Amino Acid Sequence↗
PubMed1996

cis Elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Mutational analysis of the putative leukotoxin transport genes in Actinobacillus actinomycetemcomitans.

The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces leukotoxin, a protein that specifically lyses host defense cells. The leukotoxin is similar in sequence and operon organization to the Escherichia coli alpha-hemolysin and other members of the RTX family of toxins. However, unlike the other RTX toxins, the A. actinomycetemcomitans leukotoxin is not secreted from the cell and instead remains associated with the outer membrane. Nonetheless, the A. actinomycetemcomitans Ikt operon contains two genes, IktB and IktD, that appear analagous to the toxin localization genes found in the other Gram-negative bacteria. Thus, to determine the roles of these putative transport genes in A. actinomycetemcomitans, we have used insertional mutagenesis to generate mutant strains lacking functional LktB and/or LktD. When either IktD or both IktB and IktD were inactivated, the level of detectable leukotoxin protein in the cell decreased significantly. However, the IktB and IktD mutations had no effect on the levels of leukotoxin RNA. Thus, the lack of LktB and LktD proteins must affect LktA synthesis post-transcriptionally. It is proposed that this is an indirect effect of leukotoxin mislocalization in IktB- and IktD- mutants. Finally, analysis of the mutants revealed that LktB and LktD are not essential for the formation of extracellular membrane vesicles in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

The regulation of leukotoxin production in Actinobacillus actinomycetemcomitans strain JP2.

Actinobacillus actinomycetemcomitans (A.a.) can produce a potent leukotoxin that is thought to be involved in evasion of the host immune response. In order to understand the role of A.a. and its leukotoxin in the initiation and progression of periodontal disease, it is important determine how the expression of A.a. virulence factors might be regulated by the local periodontal micro-environment. To facilitate the measurement of leukotoxin levels, a leukotoxin-beta-galactosidase gene fusion was constructed and recombined into the chromosome of A.a. strain JP2 at the leukotoxin locus. The resulting strain, AAM17, produces beta-galactosidase under control of the leukotoxin promoter. It also produces leukotoxin, since integration of the gene fusion into the chromosome was designed to produce a duplication of the leukotoxin gene. This strain was used to measure the change in leukotoxin level in response to alterations in two environmental signals: iron concentration and oxygen tension. When AAM17 was grown in iron-limited media that did not alter growth rate but did increase the levels of other iron-regulated proteins, the levels of the leukotoxin-beta-galactosidase were similar to those found in AAM17 grown in iron-replete media. These results were confirmed in strains AAM17 and JP2 by leukotoxicity assays and RNA blots. Aerobic growth of AAM17 resulted in a three-fold decrease in leukotoxin beta-galactosidase activity compared with anaerobically grown cells. These results indicate that the A.a. leukotoxin is regulated by some of the environmental signals that may vary in the gingival crevice.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Highly conserved TCR beta chain CDR3 sequences among immunodominant acetylcholine receptor-reactive T cells in murine myasthenia gravis.

In C57BL/6 (B6) mice, the T cell response to acetylcholine receptor from Torpedo californica (TAChR) provides a specific antibody response and symptoms of muscular weakness similar to those displayed in the human disease myasthenia gravis. We had found previously that the B6 T cell response to AChR shows limited clonality, both in terms of the epitopes recognized and in terms of the diversity of the TCR V beta gene segments used. We now report that all TAChR-reactive B6 T cell clones which responded to the dominant antigenic epitope and expressed the dominant TCR V beta 6 gene segment exhibited conservation of amino acid sequence in the VDJ junctional region (CDR3) of the TCR beta chain. The conserved sequence motif contained a glutamic acid residue in the CDR3 of TCR beta. Analysis of TCR beta sequences from antigen-primed lymph node cells (LNC) showed a similar enrichment for sequences having a glutamic acid in CDR3, although the overall appearance of the LNC sequences was somewhat more heterogeneous and consistent with a gradual in vitro selection of the subset of TCR found in the T cell clones. As the first example of TCR sequences in this model of myasthenia gravis, these results begin to provide a context for understanding self-non-self discrimination of AChR. In particular, the unusually conserved CDR3 sequence suggests that the conserved V beta gene utilization seen previously is directly related to recognition of the immunodominant peptide epitope in association with the experimental autoimmune myasthenia gravis-susceptibility determining MHC class II molecule I-Ab.

Amino Acid Sequence↗
PubMed1993

A panel of probes detects DNA polymorphisms in human and non-human primate isolates of a periodontal pathogen, Actinobacillus actinomycetemcomitans.

A panel of five DNA probes has been developed for use in genetic epidemiologic analysis of Actinobacillus actinomycetemcomitans, a bacterium associated with periodontal disease. Restriction fragment length polymorphism profiles were assessed using random genomic probes as well as probes for genes encoding potential virulence factors. These genotypes were compared with serotype and other phenotypic markers. Genotype was shown to be more stable than the phenotypic markers and should prove useful in epidemiological studies of periodontal disease. In addition, the use of a panel of probes has the advantage of being able to distinguish between the occurrence of a new isolate as opposed to a mutation in a strain already characterized. The panel of probes was tested on 35 isolates from humans, 18 isolates from monkeys, and two isolates from a baboon. The genetic probes revealed significant genetic diversity among the A. actinomycetemcomitans isolates. Importantly, most human isolates from an individual genotyped identically, whereas most isolates cultured from a given monkey were different, suggesting that one should exercise caution in comparison of A. actinomycetemcomitans infections in human and non-human primates.

Aggregatibacter actinomycetemcomitans↗
PubMed1991

Binding of thymic factors to the conserved decanucleotide promoter element of the T-cell receptor V beta gene is developmentally regulated and is absent in SCID mice.

The gene segments encoding the beta chain of the T-cell antigen receptor undergo rearrangement in a precise developmental order: a D beta gene segment joins to a J beta gene segment prior to the rearrangement of a V beta gene segment to join the D/J beta fusion. Current evidence suggests that the rearrangement of V beta is restricted to T cells, whereas D-to-J beta rearrangements may occur in both B and T cells. Thus, the T-cell specificity seems to be regulated by the V beta coding region or its 5' flanking sequence. In support of this hypothesis, evidence is provided for thymus-specific factors that bind a highly conserved 10-base-pair (decamer) sequence that is an essential promoter element in mouse and human V beta genes. The presence of decamer-binding activities was assayed by gel mobility-shift analysis using protein extracts from thymus, spleen, and nonlymphoid organs of adult mice. Two shifted complexes, designated T2 and T3, were seen only when the decamer was incubated with extracts from thymus. When extracts from mice of various gestational ages were tested for decamer-binding activity, one of the thymus-specific complexes, T2, was first detected at day 16; this coincides with the time of initial activation of the V beta locus. No decamer-binding activity was detected in extracts prepared from the thymuses of SCID (severe combined immunodeficiency) mice, which characteristically fail to rearrange these genes. Moreover, neither T2 nor T3 was detectable with extracts from spleen or from two T-cell lines that express the beta chain; this suggests that the presence of these two complexes is not absolutely required for transcription of the T-cell receptor beta locus. We conclude that there are tissue-specific and developmentally regulated factors that form complexes with the decamer sequence 5' of V beta; these may represent initiation factors that control the activation of germ-line T-cell receptor V beta genes for transcription and/or rearrangement.

Age Factors↗