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L L Li

Publications and source records attributed to L L Li.

At least 37 records · Page 2Linked to original sources
PubMed1995

Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.

Chronic granulomatous disease (CGD) is a recessive disorder characterized by a defective phagocyte respiratory burst oxidase, life-threatening pyogenic infections and inflammatory granulomas. Gene targeting was used to generate mice with a null allele of the gene involved in X-linked CGD, which encodes the 91 kD subunit of the oxidase cytochrome b. Affected hemizygous male mice lacked phagocyte superoxide production, manifested an increased susceptibility to infection with Staphylococcus aureus and Aspergillus fumigatus and had an altered inflammatory response in thioglycollate peritonitis. This animal model should aid in developing new treatments for CGD and in evaluating the role of phagocyte-derived oxidants in inflammation.

Alleles↗
PubMed1995

A method for monoclonal antibody isotype switching: anti-CD60 VH expression in a heavy chain-deficient hybridoma variant.

Alteration of monoclonal antibody isotype is desirable for a variety of purposes, including obtaining an improved reagent for investigative or therapeutic use. A variety of approaches for isotype switching, particularly from IgM to various IgG subclasses, have been described. Antibodies that recognize carbohydrate determinants on glycoproteins, glycolipids, or polysaccharides are generally of the IgM isotype. This includes all available antibodies to the human CD60 antigen, a determinant with cell coactivating properties described on a subset of T lymphocytes and on other cell types. In this report a new method for monoclonal antibody isotype switching is presented. A plasmid containing the VH regions of anti-CD60 linked to C gamma 1 was transfected into a spontaneously arising variant of the CD60 hybridoma that produced kappa light chain but no heavy chain. This transfected hybridoma line maintains stable production of useful quantities of IgG1 monoclonal anti-CD60 in vitro and in vivo.

Animals↗
PubMed1995

Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination.

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophilicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.

Alleles↗
PubMed1995

Expression of CD60 on multiple cell lineages in inflammatory synovitis.

BACKGROUND: CD60 is a recently described T cell subset marker that is expressed on the surface of most T lymphocytes in synovial tissue and fluid and on a smaller proportion of peripheral T cells. Activation of T lymphocytes can be triggered through CD60. CD60 is also expressed by neuroectodermally derived cells in thymic epithelium and in skin. EXPERIMENTAL DESIGN: Immunohistologic analysis of CD60 expression in synovium and thymus was performed using formalin-fixed tissue samples. Nonlymphoid cell lines grown from similar tissues were analyzed by flow cytometry. RESULTS: CD60 was readily identified in formalin-fixed, paraffin-embedded tissues. Simultaneous examination of CD60 distribution and cell morphology demonstrated that, in addition to its presence on T cells, CD60 was also expressed by a variety of nonlymphoid cells in synovium, including synovial lining cells, vascular endothelium, and dendritic-appearing cells deep within synovial tissue. Synovial tissue expression of CD60 was similar in rheumatoid arthritis and in other forms of inflammatory arthritis. In addition, it was strongly expressed by giant cells in pigmented villonodular synovitis. Surface expression of CD60 was detected by flow cytometry on cultured synoviocytes and on other CD60+ nonlymphoid cells, thus excluding adsorption of CD60 shed by T cells as a sufficient explanation of the immunohistologic findings. CONCLUSIONS: These results define the T cell-activating CD60 determinant as a broadly distributed Ag within synovial tissue, with a possible functional role in the activation of a variety of cellular populations. CD60 may also be a marker for previously undescribed cell subsets in the synovial compartment, possibly including a cell population of neuroectodermal origin.

Adult↗
PubMed1995

Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing.

OBJECTIVE: To develop and demonstrate the utility of automated DNA sequencing strategies for rapid and unambiguous identification of Mycobacterium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. DESIGN AND SPECIMENS: A 360-base pair segment of the gene (hsp65) encoding a 65-kd heat shock protein was characterized from 91 isolates assigned to 24 Mycobacterium species by traditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tuberculosis strains were also sequenced in a sample of 128 resistant organisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-positive sputum specimens from patients with tuberculosis were also studied. RESULTS: Automated DNA sequencing identified species-specific polymorphism in the target segment of hsp65, successfully identified organisms to the species level in smear-positive sputum samples, and unambiguously characterized seven genes associated with antimicrobial resistance in M tuberculosis. CONCLUSIONS: Rapid identification of M tuberculosis and other Mycobacterium species is possible by automated DNA sequencing of a portion of hsp65. The technique is also feasible for analysis of some smear-positive sputum specimens. Unambiguous characterization of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary patient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify mutations associated with antimicrobial resistance in less than 48 hours.

Alleles↗
PubMed1994

Characterization and distribution of insertion sequence IS1239 in Streptococcus pyogenes.

The human pathogenic bacterium Streptococcus pyogenes causes pharyngitis, acute rheumatic fever, glomerulonephritis and toxic-shock-like syndrome. The bacterium synthesizes several extracellular products, including the recently described streptococcal superantigen SSA, a molecule that shares considerable homology with several Staphylococcus aureus enterotoxins. While studying allelic variation at the ssa locus, six isolates expressing serotypes M4, M23, M33, M41, M43, and provisional type PT4854, were identified that had PCR products about 40-bp larger than expected, and one isolate (M15) had an amplified fragment that was more than 1-kb larger than expected. All six isolates have a 34-bp insert located 103 bp 5' of the ssa start codon. The larger product is a result of a 1110-bp insertion at the analogous location. The complementary strand of this insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to the Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least twelve copies of the sequence are present in the serotype M15 S. pyogenes isolate. This element, designated IS1239, is the first simple insertion sequence described in group-A streptococci. Results of PCR screening showed that 26 of 78 (33%) S. pyogenes isolates expressing distinct M protein serotypes contained sequences with homology to IS1239, which means that the element is widely distributed in the species.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of CD6 expression on human T lymphocytes by phorbol ester.

Activation of protein kinase C (PKC) has emerged as a major common signal-transducing mechanism for T cell activation and regulation of expression of T cell surface glycoproteins. Surface expression of the CD6 Ag is known to increase with T cell activation and CD6 itself may be involved in regulation of T cell activation. Therefore, we performed experiments to determine whether activation of PKC by phorbol ester induced an increase in CD6 expression and to investigate the mechanisms of such an effect. CD6 surface expression was up-regulated substantially in response to PMA on both mature and immature T cells, but only negligibly on B cell lines. This increase was blocked by PKC inhibitors. The PMA-induced increase in CD6 surface expression was accompanied by an increase in total CD6 protein, as detected by using Western blot analysis of whole cell lysates. After PMA stimulation, Northern blot analysis showed that steady state levels of CD6 mRNA increased in response to PMA treatment. This increase was blocked by cycloheximide, demonstrating that it was dependent on new protein synthesis. Nuclear run-on analysis showed that the rate of CD6 mRNA transcription increased by approximately two to threefold after PMA stimulation of Jurkat cells. Experiments in which we used actinomycin D showed that PMA had no significant effect on the t1/2 of CD6 mRNA. The data suggest that the effect of PMA on CD6 expression is mediated primarily by an increase in CD6 mRNA transcription after PKC activation. mAbs were used to determine whether augmented CD6 expression could be induced by perturbation of specific T cell surface molecules. Up-regulation of CD6 expression occurred when thymocytes were cultured with anti-CD2 Abs, but not with Abs to other functional T cell surface structures, and not when mature T cells were cultured with the anti-CD2 mAbs. Up-regulation of CD6 expression by activation of PKC, triggered in thymocytes by ligation of CD2, could allow CD6 to provide additional regulatory signals required for events in later stages of T cell activation and differentiation.

Antigens, CD↗
PubMed1994

Vaccination with streptococcal extracellular cysteine protease (interleukin-1 beta convertase) protects mice against challenge with heterologous group A streptococci.

Virtually all clinical isolates of group A streptococci secrete a highly conserved extracellular cysteine protease that cleaves human fibronectin and vitronectin, and converts IL-1 beta precursor to biologically active IL-1 beta. Based on the high degree of gene conservation within the species and its role in host pathogenicity, it was postulated that antibodies to the cysteine protease would confer protective immunity against S. pyogenes infection. To test this hypothesis, Swiss CD1 mice were intraperitoneally administered either saline, rabbit IgG, or IgG from rabbits immunized with the protease, and challenged with a highly virulent (minimum lethal dose approximately 10 cfu) clinical isolate of S. pyogenes expressing a heterologous cysteine protease. The results indicate that mice administered IgG from rabbits immunized with purified cysteine protease had significantly enhanced survival when compared with mice given either non-specific rabbit IgG (log rank test; chi 2; p = 0.0195) or saline (log rank test; chi 2; p = 0.0002). Moreover, mice actively immunized with the cysteine protease had a significantly longer time to death than the control group (log rank test; chi 2; p = 0.0418). The results show that the cysteine protease elicits non-type-specific immunity to challenge with heterologous S. pyogenes.

Animals↗
PubMed1994

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.

Amino Acid Sequence↗
PubMed1994

Sequencing for 4 single-copy DNA segments from human chromosome 8q24.1.

A successful method for constructing human chromosome 8q24.1 band specific probe pool by microdissection and microcloning is described in this paper. The probe pool was proved to be band-specific by fluorescence in situ hybridization. Through Southern blot analysis, 34 single copy DNA segments were obtained and four of them were sequenced. It is possible to provide useful landmarks for constructing human chromosome 8q24.1 physical map and STSs map.

Base Sequence↗
PubMed1994

Real-time molecular epidemiologic analysis of an outbreak of Streptococcus pyogenes invasive disease in US Air Force trainees.

OBJECTIVE: To determine if molecular epidemiologic techniques, including comparative automated DNA sequencing of polymorphic virulence genes, could be used in the course of a bacterial disease outbreak to unambiguously determine clonal relationships among implicated strains. DESIGN: Strains recovered from all patients with invasive infections and a sample of carriers were analyzed by multilocus enzyme electrophoresis and automated DNA sequencing of a gene encoding an extracellular protease and a highly polymorphic part of the streptokinase gene. SETTING: A US Air Force training facility in San Antonio, Tex. PATIENTS: A squadron with about 800 Air Force trainees, including three recruits with invasive Streptococcus pyogenes infections. RESULTS: Multilocus enzyme electrophoresis and automated DNA sequencing of polymorphic virulence genes unambiguously defined person-to-person spread of an otherwise rare S pyogenes clone in the course of the disease outbreak and clarified strain relationships in real time. CONCLUSIONS: Molecular strain characterization techniques can be employed rapidly in a disease outbreak to definitively resolve complex relationships among pathogenic bacteria, infer patterns of clone spread, and help formulate rational public health control measures. The approach has broad applicability to other infectious agents.

Adult↗
PubMed1993

Cleavage of interleukin 1 beta (IL-1 beta) precursor to produce active IL-1 beta by a conserved extracellular cysteine protease from Streptococcus pyogenes.

Streptococcal pyrogenic exotoxin B (SPE B), a conserved extracellular cysteine protease expressed by the human pathogenic bacterium Streptococcus pyogenes, was purified and shown to cleave inactive human interleukin 1 beta precursor (pIL-1 beta) to produce biologically active IL-1 beta. SPE B cleaves pIL-1 beta one residue amino-terminal to the site where a recently characterized endogenous human cysteine protease acts. IL-1 beta resulting from cleavage of pIL-1 beta by SPE B induced nitric oxide synthase activity in vascular smooth muscle cells and killed of the human melanoma A375 line. Two additional naturally occurring SPE B variants cleaved pIL-1 beta in a similar fashion. By demonstrating that SPE B catalyzes the formation of biologically active IL-1 beta from inactive pIL-1 beta, our data add a further dimension to an emerging theme in microbial pathogenesis that bacterial and viral virulence factors act directly on host cytokine pathways. The data also contribute to an enlarging literature demonstrating that microbial extracellular cysteine proteases are important in host-parasite interactions.

Amino Acid Oxidoreductases↗
PubMed1993

An anti-CD2 monoclonal antibody that both inhibits and stimulates T cell activation recognizes a subregion of CD2 distinct from known ligand-binding sites.

The T lymphocyte glycoprotein CD2 appears to have an important role in human T cell development and activation. A novel anti-CD2 monoclonal antibody, designed UMCD2, was shown to block E rosetting, and therefore was defined as recognizing the Tll1 ligand-binding epitope. Binding of UMCD2 to T cells and thymocytes was blocked by several, but not all, anti-Tll1 antibodies, suggesting that the Tll1 epitope consists of more than one subepitope. In functional studies, the combination of UMCD2 plus anti-Tll3 was mitogenic for T cells; in some individuals, the level of activation was as high as that seen for the combination of anti-Tll2 plus anti-Tll3. However, when UMCD2 was added to other stimuli mitogenic for T lymphocytes, such as IL-2 or anti-CD3-Sepharose, it inhibited T cell responses. Although the combination of UMCD2 and anti-Tll3 induced an increase in cytoplasmic free calcium, the inhibitory activities of UMCD2 were not accompanied by effects on calcium fluxes. A panel of previously characterized CD2 mutants was then analyzed for binding of UMCD2 and other anti-CD2 monoclonals. Surprisingly, UMCD2 bound to all mutants tested, although the other anti-CD2 antibodies with specificity for the ligand-binding region of CD2 each failed to bind to one or more mutants. These data suggest that binding of antibody to a particular CD2 epitope can have opposite effects on the state of T cell activation, depending on the costimulus. Moreover, inhibitory effects mediated through CD2 may use a signaling mechanism distinct from that used in CD2 pathway activation. Of particular interest, the portion of the CD2 ligand-binding region recognized by UMCD2 is distinct from areas of the CD2 molecule that have previously been studied.

Amino Acid Sequence↗
PubMed1993

A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin.

Streptococcus pyogenes secretes an extracellular cysteine protease that cleaves human interleukin 1 beta precursor to form biologically active IL-1 beta, a major cytokine mediating inflammation and shock. To further investigate the potential role of the cysteine protease in host-parasite interactions, the enzyme was purified to apparent homogeneity and tested for ability to degrade several human extracellular matrix proteins. Purified protease cleaved fibronectin, apparently at specific sites, and rapidly degraded vitronectin. In contrast, the protease did not have substantial activity against laminin. The cysteine protease also cleaved fibronectin from human umbilical vein endothelial cells grown in vitro. Allelic variation in the cysteine protease structural gene was studied in 67 strains expressing 39 M protein serotypes and five provisional M serologic types, and representing 50 phylogenetically distinct clones identified by multilocus enzyme electrophoresis. The gene is well conserved and allelic variation is due solely to accumulation of point mutations. Based on predicted amino acid sequences, one mature cysteine protease variant would be made by clones expressing serotypes M2, M3, M4, M5, M6, M9, M10, M11, M12, M14, M18, M22, M23, M25, M27, M41, M49, M56, M59, two provisional M types, and two clones non-typeable for M protein. Moreover, 33 of the 39 speB alleles identified encode one of three mature protease variants that differ from one another at only one or two amino acids clustered in a ten-amino acid region. All 39 alleles, and virtually all strains, encode a product that reacts with polyclonal antisera specific for purified cysteine protease. No compelling evidence was found for a primitive differentiation of the speB gene into two distinct classes, as has been proposed for M protein, opacity factor phenotype, and vir regulon architecture. The results demonstrate that the cysteine protease is well conserved in natural populations of S. pyogenes, provide additional evidence that this enzyme is involved in host-parasite interactions, and suggest that the protease plays a role in bacterial dissemination, colonization, and invasion, and inhibition of wound healing.

Alleles↗
PubMed1992

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli.

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.

Antibodies, Monoclonal↗
PubMed1992

Flanged coaxial microwave probes for measuring thin moisture layers.

We obtain an analytical, closed-form expression for the input admittance of a flanged, open-ended coaxial line radiating into a layered lossy material. The effect of higher modes as well as the dominant mode have been taken into consideration. The results obtained in the experimental measurement of the microwave reflection coefficient of the probe radiating into very thin water layers are described and compared with the results obtained from numerical calculations based on a theoretical model. The measurements were made in the 5.0-7.0 GHz range. The results demonstrate, for example, the usefulness of this type of device in measuring thin moisture layers in the outer layer of the human skin.

Body Water↗