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1.
Cancer Gene Ther ; 7(8): 1127-34, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10975673

ABSTRACT

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.


Subject(s)
HIV/immunology , Receptors, Immunologic/immunology , Recombinant Fusion Proteins/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Antigens/immunology , Humans , Molecular Sequence Data
2.
Antimicrob Agents Chemother ; 44(4): 920-8, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10722492

ABSTRACT

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.


Subject(s)
Antiviral Agents/pharmacology , HIV-1/drug effects , DNA, Viral/genetics , Drug Resistance, Microbial , Genetic Vectors , HIV-1/genetics , Humans , Microbial Sensitivity Tests , Phenotype , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction
3.
J Burn Care Rehabil ; 17(2): 117-23, 1996.
Article in English | MEDLINE | ID: mdl-8675501

ABSTRACT

Allogeneic blood transfusion (Allo/BT) and burn injury modify the cellular immune response in patients under a variety of circumstances. We designed this study to investigate the influence of Allo/BT, burn injury, and the combination of the two on in vivo natural killer (NK) cell activity in a murine model. This study demonstrated significant enhancement of in vivo NK cell activity in noninjured BALB/c mice receiving Allo/BT from C3H mice when compared to both the control and syngeneic blood transfusion group at posttransfusion day 5. When burn-injured mice were compared to sham-stressed mice, the burn-injured mice showed significant suppression of in vivo NK cell activity. Furthermore, in this strain combination model, Allo/BT modulated the suppressive effect of burn injury on in vivo NK cell activity at posttransfusion day 5 and postburn day 7.


Subject(s)
Blood Transfusion , Burns/therapy , Killer Cells, Natural/physiology , Analysis of Variance , Animals , Burns/immunology , Disease Models, Animal , Killer Cells, Natural/immunology , Male , Mice , Mice, Inbred BALB C
4.
Am J Surg ; 170(6): 558-61; discussion 561-3, 1995 Dec.
Article in English | MEDLINE | ID: mdl-7492000

ABSTRACT

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disorder of unknown pathophysiology. The role of splenectomy in the multimodality therapy of TTP is controversial. MATERIALS AND METHODS: All charts of patients with TTP at the University of Utah between 1984 and 1994 were reviewed to evaluate various treatment regimens, and specifically, the impact of splenectomy on morbidity and survival. RESULTS: Of the 15 patients identified, 14 underwent initial treatment with plasmapheresis and steroids. Nine patients were treated with medical therapy only, 6 of whom completely recovered, while 3 patients died. Six patients failed plasmapheresis and underwent splenectomy. There were no operative complications or postoperative deaths. All surgical patients had no active disease at last follow-up. CONCLUSION: Plasmapheresis and steroid administration remain the first-line therapy for TTP. This series documents that splenectomy offers excellent results with minimal morbidity and mortality in patients who do not respond to or who relapse after plasmapheresis.


Subject(s)
Purpura, Thrombotic Thrombocytopenic/therapy , Splenectomy , Adult , Combined Modality Therapy , Female , Humans , Male , Middle Aged , Plasmapheresis , Postoperative Complications , Purpura, Thrombotic Thrombocytopenic/mortality , Purpura, Thrombotic Thrombocytopenic/surgery , Retrospective Studies , Survival Rate
5.
J Biol Chem ; 264(25): 14806-11, 1989 Sep 05.
Article in English | MEDLINE | ID: mdl-2549037

ABSTRACT

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.


Subject(s)
Neoplasm Proteins/isolation & purification , Parathyroid Hormone/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Amino Acid Sequence , Animals , Cell Line , Cyclic AMP/biosynthesis , Escherichia coli/genetics , Genetic Vectors , Humans , Molecular Sequence Data , Neoplasm Proteins/physiology , Osteosarcoma , Parathyroid Hormone/physiology , Parathyroid Hormone-Related Protein , Rats , Recombinant Fusion Proteins/physiology , Tissue Plasminogen Activator/biosynthesis , Transfection
6.
Gene ; 77(1): 95-105, 1989 Apr 15.
Article in English | MEDLINE | ID: mdl-2744490

ABSTRACT

We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene (PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene (PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at -514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.


Subject(s)
Genes , Neoplasm Proteins/genetics , Promoter Regions, Genetic , Base Sequence , Blotting, Northern , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 12 , Cloning, Molecular , DNA/genetics , Exons , Humans , Introns , Molecular Sequence Data , Parathyroid Hormone/genetics , Parathyroid Hormone-Related Protein , RNA, Messenger/genetics , Sequence Homology, Nucleic Acid , Species Specificity
7.
Mol Endocrinol ; 2(12): 1176-85, 1988 Dec.
Article in English | MEDLINE | ID: mdl-2464130

ABSTRACT

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.


Subject(s)
Carrier Proteins/genetics , Cloning, Molecular , Gene Expression Regulation , Growth Hormone/physiology , Receptors, Cell Surface/genetics , Amino Acid Sequence , Base Sequence , Carrier Proteins/metabolism , Growth Hormone/pharmacology , Humans , Insulin-Like Growth Factor Binding Proteins , Molecular Sequence Data , Receptors, Cell Surface/metabolism , Receptors, Somatomedin
8.
Science ; 237(4817): 893-6, 1987 Aug 21.
Article in English | MEDLINE | ID: mdl-3616618

ABSTRACT

Humoral hypercalcemia of malignancy is a common complication of lung and certain other cancers. The hypercalcemia results from the actions of tumor factors on bone and kidney. We report here the isolation of full-length complementary DNA clones of a putative hypercalcemia factor, and the expression from the cloned DNA of the active protein in mammalian cells. The clones encode a prepro peptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino-terminal region. This previously unrecognized hormone may be important in normal as well as abnormal calcium metabolism.


Subject(s)
Hypercalcemia/genetics , Lung Neoplasms/genetics , Neoplasm Proteins/genetics , Amino Acid Sequence , Cell Line , Cloning, Molecular , DNA/genetics , Gene Expression Regulation , Humans , Lung Neoplasms/complications , Parathyroid Hormone/genetics , Parathyroid Hormone-Related Protein
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