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Biomedical subjects

T Le

Publications and source records attributed to T Le.

At least 19 recordsLinked to original sources
PubMed2000

Follicular lymphomas' BCL-2/IgH junctions contain templated nucleotide insertions: novel insights into the mechanism of t(14;18) translocation.

The human t(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between BCL-2 and D(H)/J(H) gene segments during V(D)J recombination in early B cells. De novo nucleotides are found inserted in most breakpoints and have been thus far interpreted as nontemplated N region additions. In this report, we have analyzed both direct (BCL-2/J(H)) and reciprocal (D(H)/BCL-2) breakpoints derived from 40 patients with follicular lymphoma with t(14;18). Surprisingly, we found that more than 30% of the breakpoint junctions contain a novel type of templated nucleotide insertions, consisting of short copies of the surrounding BCL-2, D(H), and J(H) sequences. The features of these templated nucleotides, including multiplicity of copies for 1 template and the occurrence of mismatches in the copies, suggest the presence of a short-patch DNA synthesis, templated and error-prone. In addition, our analysis clearly shows that t(14;18) occurs during a very restricted window of B-cell differentiation and involves 2 distinct mechanisms: V(D)J recombination, mediating the breaks on chromosome 14 during an attempted secondary D(H) to J(H) rearrangement, and an additional unidentified mechanism creating the initial breaks on chromosome 18. Altogether, these data suggest that the t(14;18) translocation is a more complex process than previously thought, involving the interaction and/or subversion of V(D)J recombination with multiple enzymatic machineries.

Base Sequence↗
PubMed2000

HLA and other host factors in transfusion-acquired HIV-1 infection.

The host and viral factors that underlie infection with HIV-1 vary considerably with some individuals progressing to AIDS within 3 to 5 years after infection, whereas others remain clinically asymptomatic for over 10 years. Host factors that may contribute to disease progression include HLA and allelic variants of the chemokine receptors CCR5 and CCR2, which have been shown to influence both long-term survival and rapid progression. In this study, we have examined the contribution of HLA and polymorphisms in CCR5 and CCR2 to long-term survival in transfusion-acquired HIV-1-infected individuals. We have found a higher number of HLA-A32 and -A25 alleles but a lower number of the HLA-B8 allele in the study group compared with the frequencies seen in the HIV-1-negative Australian caucasian population. However, there was no apparent contribution by allelic variants of CCR5 and CCR2 to long-term survival and the combined influence of HLA and CCR polymorphisms could not be evaluated in this relatively small (n = 20) group of study subjects. The results of this work support a role for HLA in long-term nonprogression though the presence in the Sydney Blood bank Cohort of nef-defective HIV-1 may confound associations between certain HLA alleles and long-term survival in the face of infection with HIV-1.

Adult↗
PubMed2000

Routine HLA-B genotyping with PCR-sequence-specific oligonucleotides (PCR-SSO) detects eight new alleles: B*0807, B*0809, B*1551, B*3529, B*3532, B*4025, B*5304 and B*5508.

This paper describes eight new alleles (B*0807, B*0809, B*1551, B*3529, B*3532, B*4025, B*5304 and B*5508) that have been found by routine HLA-B genotyping with sequence-specific oligonucleotides (SSOs). All of the new alleles have variations which cause changes in residues that occur within antigen binding pockets and T-cell recognition sites of the antigen. The new polymorphisms within these new alleles may affect the nature and specificity of peptide binding and cause differential T-cell activation, which may have an affect in transplantation.

Alleles↗
PubMed2000

Minimal catalytic domain of N-acetylglucosaminyltransferase V.

UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain.

Amino Acid Sequence↗
PubMed1999

Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in a wide variety of transformed human cells in vitro. In this study, the antitumor activity of recombinant TRAIL was analyzed in mice bearing human colon carcinoma tumors. We found that these tumors displayed a differential sensitivity to TRAIL in vivo that paralleled their susceptibility to TRAIL-induced apoptosis in vitro. Treatment of TRAIL-sensitive tumors 3 days after tumor challenge resulted in a dose-dependent inhibition of growth and the elimination of tumors in many mice. Colon carcinoma cell lines could be further sensitized to TRAIL-induced apoptosis in vitro by the addition of the chemotherapeutic agent camptothecin. Moreover, the combination of TRAIL and CPT-11, a water-soluble analogue of camptothecin, greatly enhanced the antitumor activity of TRAIL in vivo. TRAIL plus CPT-11 treatment of both 3- and 10-day established TRAIL-sensitive tumors resulted in both a significant inhibition of tumor growth and a high proportion of complete tumor regressions. Treatment of TRAIL-resistant tumors with TRAIL and CPT-11 dramatically slowed tumor growth and induced a transient tumor regression. These data demonstrate that TRAIL alone is a potent antitumor agent in vivo, and its activity can be significantly enhanced in combination with the chemotherapeutic agent CPT-11.

Animals↗
PubMed1999

Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems.

Synthetic CD8+ cytotoxic T-lymphocyte (CTL) peptide epitope based vaccines are being developed against a number of human diseases. Here we describe extensive preclinical testing of peptide epitope vaccines formulated with a protein as a source of CD4 help and Montanide ISA 720, an adjuvant currently in human clinical trials. Such water-in-oil formulations could effectively co-deliver several peptide epitopes and simultaneously induce multiple independent CTL responses. The efficiency of CTL induction by some peptides was, however, dependent on the aqueous buffer conditions, with poor performance correlating with non-covalent peptide oligomerisation. Any of a number of proteins currently used in human vaccines could supply CD4 help and no difference in CTL induction was obtained if the CD4 response was amnestic or a primary. Peptide immunisation was found to induce long term CTL memory and the recall of protective responses did not depend on an amnestic CD4 response. Slow pyroglutamic acid formation and rapid oxidation of methionine residues was observed in water-in-oil formulations, however, the latter had no effect on CTL induction. These data highlight the need to monitor for potential deleterious chemical events and interpeptide interactions, but illustrate that peptide based vaccination can effectively deliver multiple epitopes, in conjunction with any protein, and induce protective memory.

Adjuvants, Immunologic↗
PubMed1999

Plasmid DNA malaria vaccine: tissue distribution and safety studies in mice and rabbits.

To evaluate the safety of a plasmid DNA vaccine, tissue distribution studies in mice and safety studies in mice and rabbits were conducted with VCL-2510, a plasmid DNA encoding the gene for the malaria circumsporozoite protein from Plasmodium falciparum (PfCSP). After intramuscular administration, VCL-2510 plasmid DNA was detected initially in all of the highly vascularized tissues, but at later time points was found primarily in the muscle at the site of injection, where it persisted for up to 8 weeks. After intravenous administration, plasmid DNA initially distributed at a relatively low frequency to all the tissues examined except the gonads and brain. However, plasmid DNA rapidly cleared, and by 4 weeks postadministration could be detected only in the lung of one of six animals evaluated. In a safety study in mice, eight repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 1, 10, and 100 microg had no adverse effects on clinical chemistry or hematology, and did not result in any organ pathology or systemic toxicity. In a safety study in rabbits, six repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 0.15 and 0.45 mg had no discernible effects on clinical chemistry, hematology, or histopathology. No evidence of autoimmune-mediated pathology, anti-nuclear antibodies (ANA), or antibodies to dsDNA were observed in the mouse or rabbit studies.

Age Factors↗
PubMed1999

Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection.

Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.

Animals↗
PubMed1999

Frequent deregulation of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma.

Alterations of the p16 gene in neuroblastoma are very rare. Pronounced expression of p16 at both the transcript and protein levels, however, was observed in 7 of 19 (39%) neuroblastoma cell lines and 2 of 6 (33%) primary neuroblastoma samples. As p16 expression is tightly controlled in a feedback loop with Rb, we investigated the possibility that changes in p16 expression were reflective of alterations of the downstream components in the G1 regulatory pathway. Two cell lines and one primary sample highly expressing p16 were shown to harbor CDK4 amplification. The cyclin D2 gene was infrequently expressed in neuroblastoma cell lines and did not correlate with p16 expression. Slight variations in the expression of CDK6, cyclins D1, D3 and E; and E2F1 and E2F2 among the cell lines were observed, without apparent correlation with p16 status. No mutations to the p16-binding site of CDK4 and CDK6 nor any mutations to the coding region of p16 itself were identified in neuroblastoma cell lines. Despite frequent N-myc amplification in these cell lines, no relationship with this gene was observed either. All cell lines contained Rb protein with varying degrees of phosphorylation, which bears no correlation with p16 expression. Overall, alterations of the G1 pathway in neuroblastoma included relatively frequent p16 expression and infrequent CDK4 amplification and cyclin D2 expression. Despite a reported feedback relationship between p16 expression and Rb/G1 deregulation, p16 expression in neuroblastoma cell lines is independent of Rb gene and phosphorylation status and, in contrast to other cell lines where expression of p16 leads to G1/S arrest, neuroblastoma cell lines proliferate in the presence of elevated levels of p16.

Binding Sites↗
PubMed1999

Clinically apparent early stage invasive epithelial ovarian carcinoma: should all be treated similarly?

OBJECTIVES: The role of adjuvant therapy in patients with early stage ovarian carcinoma has not been clearly defined. Most randomized trials examining this issue have not used the vigorous staging exploration accepted as today's standard. This report examines the natural history of patients after surgically documented stage 1 ovarian carcinoma followed expectantly. METHODS: A retrospective chart review was carried out using strict criteria to include only patients who had an adequate staging procedure performed by gynecologic oncologists following a fixed protocol from 1987 to 1997. Patients' demographic data as well as current disease status were abstracted and analyzed. RESULTS: A total of 80 comprehensive surgical staging procedures were carried out over a 10-year period for apparent stage 1 ovarian cancer at the time of exploratory laparotomy. Fifty cases were true surgicopathological stage 1. It was found that serous and anaplastic tumors were more likely than other subtypes to be upstaged by the procedure. Further follow-up confirmed the excellent prognosis of early stage serous, endometrioid, and mucinous tumor with only one recurrence noted in an extraabdominal location in a patient with serous histology with no postoperative adjuvant therapy. Clear cell histology stands out as a significant recurrence risk (33%) despite an initially negative surgical assessment. CONCLUSION: Careful surgical exploration can identify a group of patients with early stage epithelial ovarian carcinoma who will benefit little from further adjuvant therapy. Patients with clear cell histology prove to be at a high risk for recurrence even at an early stage such that chemotherapy should be considered.

Adult↗
PubMed1999

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Acyltransferases↗
PubMed1999

Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.

To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.

Animals↗
PubMed1999

Indomethacin-induced gastric antral damage in hamsters: are neutrophils involved?

BACKGROUND: A direct role for neutrophils in the pathophysiology of indomethacin-induced gastric damage is controversial. Therefore, such damage was evaluated in hamsters. METHODS: Gastric antral damage was evaluated 4 h after the oro-gastric administration of indomethacin (30 mg/kg). Prior to indomethacin, hamsters were treated with various pharmacological agents: rebamipide, methotrexate or anti-neutrophil serum (ANS). The number of circulating neutrophils was determined from Wright-Giemsa stained blood smears. Myeloperoxidase (MPO) activity was measured as a marker of gastric antral neutrophil infiltration. RESULTS: Indomethacin caused primarily gastric antral damage. By histology, this damage did not penetrate the muscularis mucosa. A significant increase in gastric antral MPO activity was also found in indomethacin-treated hamsters. Rebamipide decreased macroscopic gastric antral damage in a dose-related fashion. Methotrexate treatment reduced the circulating blood neutrophil number by 38-44%, but did not affect gastric damage. ANS treatment resulted in near complete neutropenia, and also in a substantial reduction (84%) in gastric antral MPO activity. However, gastric antral damage was not significantly altered by ANS. CONCLUSIONS: Neutrophils are not directly involved in the pathophysiology of indomethacin-induced damage to the hamster gastric antrum.

Alanine↗
PubMed1999

Effect of naproxen on the hamster gastric antrum: ulceration, adaptation and efficacy of anti-ulcer drugs.

BACKGROUND: Various animal models of non-steroidal anti-inflammatory drug (NSAID)-induced gastric ulceration exist. These models have limitations, which make them less relevant to the human situation. AIM: : To develop a more simple and more relevant model of NSAID-induced gastric ulceration and adaptation. METHODS: Gastric ulceration was evaluated following the orogastric administration of naproxen (80 mg/kg b.d.) to hamsters. The effects of misoprostol and famotidine on gastric acid secretion and ulceration were also determined. Gastric adaptation was evaluated by proliferating cell nuclear antigen (PCNA) immunohistochemistry, in hamsters given naproxen for 3 weeks. Antral resistance to acute injury by NSAIDs and ethanol was also determined in these animals. RESULTS: Naproxen caused primarily gastric antral ulceration, which decreased from day 3 to day 21. This gastric adaptation was accompanied by an increase in PCNA positive cells, particularly on days 7 and 14. The adapted gastric antral mucosa was resistant to acute damage by various agents. Misoprostol (1 or 100 microg/kg) prevented antral ulceration, without affecting gastric acid secretion. Despite decreasing acid output by> 90%, famotidine (30 mg/kg) failed to prevent ulceration. CONCLUSION: The administration of naproxen (80 mg/kg b.d.) to hamsters is a simple, reliable and relevant method for evaluating NSAID-induced gastric antral ulceration and adaptation.

Adaptation, Physiological↗
PubMed1999

The spectrum of mutations in TBX3: Genotype/Phenotype relationship in ulnar-mammary syndrome.

Ulnar-mammary syndrome (UMS) is a pleiotropic disorder affecting limb, apocrine-gland, tooth, hair, and genital development. Mutations that disrupt the DNA-binding domain of the T-box gene, TBX3, have been demonstrated to cause UMS. However, the 3' terminus of the open reading frame (ORF) of TBX3 was not identified, and mutations were detected in only two families with UMS. Furthermore, no substantial homology outside the T-box was found among TBX3 and its orthologues. The subsequent cloning of new TBX3 cDNAs allowed us to complete the characterization of TBX3 and to identify alternatively transcribed TBX3 transcripts, including one that interrupts the T-box. The complete ORF of TBX3 is predicted to encode a 723-residue protein, of which 255 amino acids are encoded by newly identified exons. Comparison of other T-box genes to TBX3 indicates regions of substantial homology outside the DNA-binding domain. Novel mutations have been found in all of eight newly reported families with UMS, including five mutations downstream of the region encoding the T-box. This suggests that a domain(s) outside the T-box is highly conserved and important for the function of TBX3. We found no obvious phenotypic differences between those who have missense mutations and those who have deletions or frameshifts.

Abnormalities, Multiple↗
PubMed1999

Identification of survival motor neuron as a transcriptional activator-binding protein.

Spinal muscular atrophy (SMA) is an inherited neuro-muscular disease characterized by specific degeneration of spinal cord anterior horn cells and subsequent muscle atrophy. Survival motor neuron ( SMN ), located on chromosome 5q13, is the SMA-determining gene. In the nucleus, SMN is present in large foci called gems, the function of which is not yet known, while cytoplasmic SMN has been implicated in snRNP biogenesis. In SMA patients, SMN protein levels and the number of gems generally correlate with disease severity, suggesting a critical nuclear function for SMN. In a screen for proteins associated with the nuclear transcription activator 'E2' of papillomavirus, two independent SMN cDNAs were isolated. The E2 and SMN proteins were found to associate specifically in vitro and in vivo. Expression of SMN enhanced E2-dependent transcriptional activation, and patient-derived SMN missense mutations reduced E2 gene expression. Our results demonstrate that SMN interacts with a nuclear transcription factor and imply that SMN may serve a role in regulating gene expression. These observations suggest that SMA may in part result from abnormal gene expression and that E2 may influence viral gene expression through SMN interaction.

Cyclic AMP Response Element-Binding Protein↗
PubMed1999

Endogenous glucocorticoids released during acute toxic liver injury enhance hepatic IL-10 synthesis and release.

Endogenous glucocorticoids are known to play a role in the regulation of the inflammatory response possibly by modulating pro- and anti-inflammatory cytokine expression. We examined endogenous glucocorticoid secretion, hepatic damage, tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10) mRNA expression and release in rats treated with carbon tetrachloride (CCl4) after treatment with vehicle or a glucocorticoid receptor antagonist (RU-486). Rats treated with CCl4 demonstrated striking elevations of plasma corticosterone levels. Inhibition of endogenous glucocorticoid activity by pretreatment with the glucocorticoid receptor antagonist RU-486 resulted in augmented CCl4-mediated hepatotoxicity, as reflected by histology and serum transaminase levels, which were independent of alterations in serum TNF-alpha levels or hepatic mRNA expression. CCl4 treatment resulted in enhanced hepatic IL-10 mRNA expression and elevated serum IL-10 levels, which were markedly attenuated by glucocorticoid receptor blockade. In summary, significant endogenous glucocorticoid release occurs during acute toxic liver injury in the rat and suppresses the inflammatory response independent of effects on TNF-alpha but possibly by upregulating hepatic IL-10 production.

Acute Disease↗