Determination of residual antiplatelet activity of clopidogrel before neuraxial injections.

BACKGROUND: Guidelines recommend discontinuation of clopidogrel for 7 days before a neuraxial injection, while other directives suggest that 5 days might be adequate. We examined the time course of antiplatelet activity after clopidogrel discontinuation in patients undergoing epidural injections. METHODS: Thirteen patients were studied at baseline, 3, 5, and 7 days after discontinuation of clopidogrel. P(2)Y(12) determinations were performed using the VerifyNow(®) assay (Accumetrics, San Diego, CA, USA), and clot closure times with stimulation by collagen/epinephrine and collagen/adenosine diphosphate using the PFA-100(®) (Platelet Function Analyzer, Siemens Diagnostics, Deerfield, IL, USA). Repeated-measures ANOVA was used to evaluate P(2)Y(12) platelet reaction units, PFA-100 closure times, and per cent P(2)Y(12) inhibition values. Wilcoxon's signed-rank test was used to compare the frequencies of ≥30%, 11-29%, and ≤10% platelet inhibition between the baseline and subsequent sampling points after discontinuation of clopidogrel. RESULTS: On day 3 after clopidogrel discontinuation, two subjects had ≥30%, seven subjects had 11-29%, and four subjects had ≤10% platelet inhibition; the corresponding numbers were 0, 3, and 10 subjects on day 5 (P=0.04). There were no differences between the ≥30%, 11-29%, and <10% platelet inhibition groups between days 5 and 7 (0, 0, and 13 subjects, P=1.0). PFA-ADP closure times were normal throughout the study period except in one patient. CONCLUSIONS: These findings support the recommendation that discontinuation of clopidogrel for 5 days allows >70% of platelet function and might be adequate before a neuraxial injection is performed.

Methods of cytologic smear preparation and fixation. Effect on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3.

OBJECTIVE: To evaluate the effect of fixation and methods of cytologic smear preparation on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3. STUDY DESIGN: Scrape cytology smears and formalin-fixed, paraffin-embedded tissue sections (FPTS) of 20 unfixed, fresh specimens submitted for intraoperative consultation were studied by the immunoperoxidase method. In addition to the morphologic examination, the smears and FPTS were evaluated for intensity and proportion scores. For each specimen, two scrape cytology smears were wet fixed in 95% ethanol, and 12 smears were air dried without fixation. Air-dried smears were either postfixed after rehydration in saline or fixed directly without rehydration by one of the three fixatives: alcoholic formalin, 95% ethanol with 5% acetic acid or 95% ethanol. RESULTS: Both intensity and proportion scores were higher with rehydrated, air-dried smears as compared to those without rehydration and were comparable to those with wet-fixed smears and FPTS. In the rehydrated group, the optimum results were achieved when the smears were postfixed with alcoholic formalin. CONCLUSION: The method of preparation and fixation had variable effects on the immunoreactivity of anticytokeratin antibody AE1/AE3. The optimum results were achieved with saline-rehydrated, air-dried smears post-fixed in alcoholic formalin. To evaluate the role of inter-sample variation, further, larger studies are recommended on this and other antibodies before applying them to different types of cytologic smears.

The role of constitutive NF-kappaB activity in PC-3 human prostate cancer cell invasive behavior.

The purpose of this study was to determine if increased NF-kappaB activity of highly invasive PC-3 cells contributed to their invasive behavior. Increased NF-kappaB activity has been observed in several malignant tumors and it may have an important role in tumorigenesis, progression and chemotherapy resistance. By serial selection, we obtained invasion variant PC-3 cell sublines. The PC-3 High Invasive cells invade readily through a Matrigel reconstituted basement membrane while PC-3 Low Invasive cells have low baseline invasion activity. In these studies, we discovered that NF-kappaB DNA binding activity was increased in PC-3 High Invasive cells when compared to PC-3 Low Invasive cells by electrophoretic mobility shift assay (EMSA). Gel supershift assays showed a 4-fold increase in p65 containing complexes and a 2.2-fold increase in the p50 containing complexes in the PC-3 High Invasive cells. Luciferase reporter assays showed that NF-kappaB dependent transcription activity was increased 10.2 +/- 2.5-fold in the highly invasive cells (P < 0.002). The PC-3 High Invasive cells showed a constitutive increase in phospho-IkappaB alpha and introduction of the super-repressor IkappaB alpha S32/36A inhibited NF-kappaB activity to 19.2 +/- 2.5 percent of control transfected cells (P < or = 0.001). The IkappaBa super-repressor reduced the basement membrane invasion of PC-3 High Invasive cells from 6.2 +/- 1.1 to 3.8 +/- 0.4 percent (P < 0.002) with no decrease in cell viability or proliferation. These results demonstrate that increased NF-kappaB activity contributed directly to the invasive behavior of PC-3 High Invasive prostate cancer cells.

Prostate-specific antigen expression and lipochrome pigment granules in the differential diagnosis of prostatic adenocarcinoma versus seminal vesicle-ejaculatory duct epithelium.

BACKGROUND: Lipochrome pigment granules (LPGs) and prostate-specific antigen (PSA) localization have been cited as helpful adjuncts in differentiating atypical histologic patterns of seminal vesicle-ejaculatory duct (SVED) from prostatic adenocarcinoma. However, LPGs have been described in both benign and neoplastic prostatic acini, and PSA expression within the intraprostatic SVED has not been fully explored. DESIGN: Fifty radical prostatectomy specimens were studied for LPGs and 9 cases for PSA expression. RESULTS: Two morphologic types of LPGs (type 1 and type 2) were observed. The reproducibility in classifying LPGs was evaluated by kappa statistics, which demonstrated a strong agreement between 4 observers. Type 1 was restricted to SVED in all 50 specimens. Type 2 was subclassified into 2A and 2B. Type 2 LPGs were observed in prostatic acini of different zones, high-grade prostatic intraepithelial neoplasia, prostatic adenocarcinoma, and occasionally with type 1 LPG in SVED. Focal reactivity for PSA in the distal portion of SVED near urethra was noted in 1 of 9 cases. CONCLUSION: Awareness about morphologic differences between the 2 types of LPGs could help to avoid a potential diagnostic pitfall of misinterpreting SVED epithelium for adenocarcinoma. Caution is recommended in interpreting PSA expression, since rare focal PSA reactivity was observed in the distal SVED.

The retinoblastoma gene product (Rb) induces binding of a conformationally inactive nuclear factor-kappaB.

Nuclear factor-kappaB (NF-kappaB) regulates expression of several viral and cellular genes including the human immunodeficiency virus long terminal repeat, major histocompatibility complex class I, and interleukin 2Ralpha cytokine genes. Here we report that the retinoblastoma gene product (Rb) stimulates binding of the NF-kappaB p50 homodimer. The addition of Rb protein to an in vitro gel shift binding assay stimulated p50 binding greater than 10-fold. Interestingly, by analyzing NF-kappaB-dependent transcription activity in vitro, we demonstrate that Rb suppresses transcriptional activity of p50. Chymotrypsin analysis suggests that Rb induces a conformational change in the NF-kappaB-DNA complex, resulting in binding of a transcriptionally inactive complex. Finally, we demonstrate by coimmunoprecipitation analysis that the Rb-p50 complex is present in Jurkat cell extracts. Our results suggest that Rb may play an important role in regulation of NF-kappaB transcriptional activity.

Human T-cell lymphotropic virus type 1 Tax1 activation of NF-kappa B: involvement of the protein kinase C pathway.

Human T-cell lymphotropic virus type 1 Tax1 induces the activation and nuclear localization of the cellular transcription factor, NF-kappa B. Treatment of cells with calphostin C, a protein kinase C (PKC) inhibitor, blocked induction of NF-kappa B DNA binding activity in human T-cell lymphotropic virus type 1-transformed C81 cells and Tax1-stimulated murine pre-B cells, suggesting that PKC was an important intermediate in the NF-kappa B induction pathway. We further demonstrate that Tax1 associates with, and activates, PKC. PKC was coimmunoprecipitated with anti- Tax1 sera from Tax1-expressing MT4 extracts and Jurkat extracts in the presence of exogenous Tax1 protein. In addition, the glutathione-S-transferase-Tax1 protein bound specifically to the alpha, delta, and eta PKC isoenzymes synthesized in rabbit reticulocyte lysates. The addition of Tax1 to in vitro kinase reaction mixtures leads to the phosphorylation of Tax1 and an 18-fold increase in the autophosphorylation of PKC. Transfection of Jurkat cells with wild-type Tax1 stimulated membrane translocation of PKC. In contrast, Tax1 mutant M22, which fails to stimulate NF-kappa B-dependent transcription, failed to stimulate membrane translocation of PKC. Tax1 did not directly increase PKC phosphorylation of I kappa B alpha. Our results are consistent with a model in which Tax1 interacts with PKC and stimulates membrane translocation and triggering of the PKC pathway. Subsequent steps in the PKC cascade likely stimulate phosphorylation of I kappa B alpha.

Stabilization of wild-type p53 in human T-lymphocytes transformed by HTLV-I.

Adult T-cell leukemia/lymphoma is an aggressive malignancy associated with infection by the human T-lymphotropic virus type-I (HTLV-I). We now demonstrate that p53 expression is elevated in the HTLV-I-transformed T-lymphocyte lines C81, MT-2, MT-4 and HUT 102. In pulse-chase experiments, the p53 protein demonstrated a prolonged half-life of 2 to 8 h in HTLV-I-transformed cells compared with 0.5 to 1.0 h for wild-type p53 in primary human and murine fibroblasts, or human peripheral blood lymphocytes. In cell lines C81 and HUT 102, which exhibited the longest p53 protein half-life, the wild-type-related PAb1620 epitope was detected at reduced levels. The PAb240 mutant-related p53 epitope was not detected in any of the transformed cell lines. By direct sequence analysis of RT-PCR products, the entire p53 cDNA coding sequence was determined to be wild-type in all four cell lines. Stabilization of wild-type p53 may represent its functional inactivation and contribute to lymphocyte transformation by HTLV-I.

Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression.

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription probably play an important role in initiation and maintenance of virus replication. We have identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]) at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. The basal promoter strength of constructs that contained deletions in the R/U5 region of the HTLV-I long terminal repeat were analyzed by chloramphenicol acetyltransferase assays following transfection of Jurkat T cells. We consistently observed a 10-fold decrease in basal promoter activity when sequences between +202 to +246 were deleted. By reverse transcriptase polymerase chain reaction RNA analysis, we confirmed that the drop in chloramphenicol acetyltransferase activity was paralleled by a decrease in the level of steady-state RNA. DRE 1 did not affect the level of Tax1 transactivation. Using a gel shift assay, we have purified a highly enriched fraction that could specifically bind DRE 1. This DNA affinity column fraction contained four detectable proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: p37, p50, p60, and p100. The affinity column fraction stimulated HTLV-I transcription approximately 12-fold in vitro. No effect was observed with the human immunodeficiency virus or adenovirus major late promoters. Following renaturation of the proteins isolated from an SDS-containing gel, p37, but not the other protein fractions, was able to specifically bind to DRE 1.

Extracellular Tax1 protein stimulates tumor necrosis factor-beta and immunoglobulin kappa light chain expression in lymphoid cells.

The human T-cell leukemia virus type I tax1 gene product is responsible for the increased expression of several cytokine and cellular genes that contain NF-kappa B regulatory sequences. Our laboratory has previously demonstrated that purified, extracellular Tax1 protein induced the nuclear accumulation of NF-kappa B binding activity in lymphoid cells. Since HTLV-I infection causes increased levels of lymphotoxin tumor necrosis factor-beta [TNF-beta] and immunoglobulin secretion, we have studied the interaction of NF-kappa B proteins from Tax1-stimulated cells with the TNF-beta and immunoglobulin kappa (Ig kappa) light chain genes. Tax1 induction of NF-kappa B occurred in the presence of cycloheximide, and Tax1 stimulation did not result in increased levels of NF-kappa B or c-rel RNA. These results indicate that new synthesis of NF-kappa B proteins was not required for induction of NF-kappa B-binding activity. With use of the Ig kappa NF-kappa B-binding site as a probe, two distinct NF-kappa B gel shift complexes were induced by the Tax1 protein. A slower-migrating complex, C1, was inhibited by the addition of purified I kappa B. In contrast, the faster-migrating C2 complex was not inhibited by I kappa B, but C2 was increased by detergent treatment of cytoplasmic extracts, suggesting that its binding activity was also regulated by an inhibitor. The Tax1-stimulated proteins that interacted with the NF-kappa B-binding sites in the Ig kappa and TNF-beta promoters were distinct. A 75-kDa protein preferentially associated with the Ig kappa NF-kappa B-binding site. In contrast, a 59-kDa protein associated with the TNF-beta NF-kappa B-binding site. Tax1 stimulation led to increased levels of TNF-beta and Ig kappa mRNA, as measured by reverse transcription and polymerase chain reaction analysis. These results represent the first experimental evidence that extracellular Tax1 can regulate the expression of endogenous cellular genes.

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.

Transdominant human T-cell lymphotropic virus type I TAX1 mutant that fails to localize to the nucleus.

Human T-cell lymphotropic virus type I (HTLV-I) encodes a 40-kDa nuclear transactivating phosphoprotein, TAX1. The results presented in this study demonstrate that deletion of amino acids 2 through 59 of TAX1 (delta 58 TAX1) decreased transactivation of the HTLV-I long terminal repeat 10- to 20-fold. S1 nuclease analysis revealed that the decrease in transactivation of the HTLV-I long terminal repeat was associated with a lack of RNA synthesis. In contrast to the nuclear localization of the wild-type TAX1 protein, indirect immunofluorescence analysis demonstrated that delta 58 TAX1 failed to localize to the nucleus, indicating that the TAX1 nuclear localization sequence is present in amino acids 2 through 59. Cotransfection of wild-type and mutant TAX1 DNAs resulted in the cytoplasmic accumulation of TAX1 and a 25-fold decrease in transactivation. Although several possibilities which may account for this transdominant effect exist, we favor a model in which delta 58 TAX1 interferes with the nuclear localization of wild-type TAX1 protein, perhaps by forming heterodimer complexes.

Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax1 expressed in E. coli.

A protocol which involves sequential ammonium sulfate precipitation and zinc chelate chromatography to purify the HTLV-I Tax1 protein expressed in E. coli is described. The final Tax1 product is greater than 90% pure and the yield is approximately 1 mg per liter of liquid culture. The purified Tax1 protein is biologically active in indirect in vitro DNA binding assays and cellular NF-kB induction experiments.

Induction of nuclear NF-kappa B DNA binding activity after exposure of lymphoid cells to soluble tax1 protein.

We demonstrate that purified HTLV-I Tax1 protein can be taken up by 70Z/3 lymphoid cells and localized in both the nuclear and cytoplasmic compartments. Introduction of the Tax1 protein into the growth medium of 70Z/3 cells resulted in the rapid and transient induction of NF-kappa B binding activity in the nuclear fraction. Tax1 activation of NF-kappa B was not sensitive to either staurosporin or prolonged stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, suggesting that Tax1-dependent NF-kappa B activation did not require the protein kinase C pathway. Purified Tax1 did not directly increase NF-kappa B binding activity in 70Z/3 cytoplasmic extracts, suggesting that NF-kappa B induction may require cellular factors. Western blot and competitive radioimmunoassays demonstrated that Tax1 protein was present in the tissue culture media of HTLV-I-transformed cell lines. These results show that extracellular Tax1 may regulate cellular gene expression in noninfected cells.

A 36-kilodalton cellular transcription factor mediates an indirect interaction of human T-cell leukemia/lymphoma virus type I TAX1 with a responsive element in the viral long terminal repeat.

The human T-cell leukemia/lymphoma virus type I (HTLV-I) trans activator, TAX1, interacts indirectly with a TAX1-responsive element, TRE-2, located at positions -117 to -163 in the viral long terminal repeat. This report describes the characterization of a 36-kilodalton (kDa) protein identified in HeLa nuclear extract which mediates the interaction of TAX1 with TRE-2. Purification of the protein was achieved by zinc chelate chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The renatured 36-kDa protein bound specifically to a TRE-2 oligonucleotide but not to nonfunctional base substitution mutant probes in a gel retardation assay. Renatured proteins of differing molecular weights were unable to form this complex. In addition, the 36-kDa protein specifically activated transcription from the HTLV-I promoter in vitro. Purified TAX1 protein formed a complex with the TRE-2 oligonucleotide in the presence of the 36-kDa protein, suggesting that indirect interaction of TAX1 with the viral long terminal repeat may be one of the mechanisms by which HTLV-I transcription is regulated.

Isolated cardiac amyloidosis associated with sudden death.

An unusual case of localized cardiac amyloidosis was associated with sudden death in the absence of prior symptoms of heart disease. Autopsy studies showed amyloid deposition in the ventricular myocardium and conduction system; there were no deposits in other viscera, and evidence of a plasma cell dyscrasia was lacking. Thus, it appears that isolated involvement of the heart by amyloidosis may cause death, even in patients who do not have a history of congestive heart failure or arrhythmia.