The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway.

Flavopiridol (FP), an inhibitor of cyclin dependent kinases 1, 2 and 4, potently induced apoptosis in U937 human monoblastic leukemia cells. This process was accompanied by characteristic morphological changes, inner mitochondrial membrane permeability transition, release of cytochrome c, processing of procaspases, and generation of reactive oxygen species. Significantly, the general caspase inhibitor Boc-FMK did not block the release of cytochrome c, whereas it did block cleavage of BID and the loss of Deltapsi(m). Neither FP-induced apoptosis nor cytochrome c release was inhibited by the pharmacological caspase-8 inhibitor IETD-FMK or endogenous expression of viral caspase-8 inhibitor CrmA. Finally, FP-mediated apoptosis, but not cytochrome c release, was partially blocked by the free radical scavenger LNAC. Collectively, these findings indicate that FP induces apoptosis in U937 cells via the release of cytochrome c from the mitochondria and independently of activation of procaspase-8.

The cyclin-dependent kinase inhibitor (CDKI) flavopiridol disrupts phorbol 12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells.

Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiridol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 human leukemia cells in relation to differentiation and apoptosis. Simultaneous, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA significantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribose) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhanced apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline in leukemic cell clonogenicity. Moreover, PMA/FP cotreatment significantly increased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration of FP blocked PMA-induced expression and reporter activity of the CDKI p21WAF/CIP1 and triggered caspase-mediated cleavage of the CDKI p27KIP1. Coexposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase 1 inhibitor U0126 did not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephosphorylation of the retinoblastoma protein (pRb), an event followed by pRb cleavage culminating in the complete loss of underphosphorylated pRb (approximately Mr 110,000) by 24 h. Finally, gel shift analysis revealed that coadministration of FP with PMA for 8 h led to diminished E2F/pRb binding compared to the effects of PMA alone. Collectively, these findings indicate that FP modulates the expression/activity of multiple signaling and cell cycle regulatory proteins in PMA-treated leukemia cells and that such alterations are associated with mitochondrial damage and apoptosis rather than maturation. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic strategy.

Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53.

Determinants of differentiation and apoptosis in myelomonocytic leukemia cells (U937) exposed to the novel hybrid polar compound SAHA (suberoylanilide hydroxamic acid) have been examined. In contrast to hexamethylenbisacetamide (HMBA), SAHA-related maturation was limited and accompanied by marked cytoxicity. SAHA-mediated apoptosis occurred within the G0G1 and S phase populations, and was associated with decreased mitochondrial membrane potential, caspase-3 activation, PARP degradation, hypophosphorylation/cleavage of pRB, and down-regulation of c-Myc, c-Myb, and B-Myb. Enforced expression of Bcl-2 or Bcl-XL inhibited SAHA-induced apoptosis, but only modestly potentiated differentiation. While SAHA induced the cyclin-dependent kinase inhibitor p21CIP1, antisense ablation of this CDKI increased, rather than decreased, SAHA-related lethality. In contrast, conditional expression of wild-type p53 failed to modify SAHA actions, but markedly potentiated HMBA-induced apoptosis. Finally, SAHA modestly increased expression/activation of the stress-activated protein kinase (SAPK/JNK); moreover, SAHA-related lethality was partially attenuated by a dominant-negative c-Jun mutant protein (TAM67). SAHA did not stimulate mitogen-activated protein kinase (MAPK), nor was lethality diminished by the specific MEK/MAPK inhibitor PD98059. These findings indicate that SAHA potently induces apoptosis in human leukemia cells via a pathway that is p53-independent but at least partially regulated by Bcl-2/Bcl-XL, p21CIP1, and the c-Jun/AP-1 signaling cascade.

Frequency and significance of antibodies to asialoglycoprotein receptor in type 1 autoimmune hepatitis.

Antibodies to asialoglycoprotein receptor have diagnostic specificity for autoimmune hepatitis, but it is uncertain if they are complementary or redundant markers of the disease. Our aims were to assess their frequency and significance in type 1 autoimmune hepatitis and determine their contribution to the evaluation of these patients. Sera from 54 well-characterized patients were evaluated for antibodies to asialoglycoprotein receptor by a radioimmunofiltration assay based on rabbit-derived protein. Forty-four patients (82%) were seropositive. Seropositive patients were distinguished from seronegative counterparts by having higher serum gamma globulin (3.7 +/- 0.2 g/dl vs 2.3 +/- 0.3 g/dl, P = 0.0007) and immunoglobulin G levels (3707 +/- 179 mg/dl vs 2203 +/- 263 mg/dl, P = 0.0005) at presentation and a greater frequency of relapse after drug withdrawal (88% vs 33%, P = 0.01). Seropositivity for smooth muscle and/or antinuclear antibodies did not define treatment outcomes and antinuclear antibodies occurred less frequently than the other markers. Concurrent testing for antibodies to asialoglycoprotein receptor and smooth muscle identified all patients. We conclude that antibodies to asialoglycoprotein receptor are common in type 1 autoimmune hepatitis and they identify patients with a high frequency of relapse after corticosteroid withdrawal. Concurrent testing for these antibodies and smooth muscle antibodies has the same diagnostic sensitivity as testing for antinuclear and smooth muscle antibodies but a greater prognostic implication.

Immunoglobulin M reactivity towards the immunologically active region sp75 of the core protein of hepatitis C virus (HCV) in chronic HCV infection.

Serum samples from a cohort of patients with chronic hepatitis C virus (HCV) infection were assayed for IgM anti-HCV/core reactivity with a "site-specific" ELISA, in which the solid phase was charged with the synthetic polypeptide analogue corresponding to the first 75 amino acids of the HCV core antigen (sp75). Thirteen of 24 (54%) patients exhibited IgM anti-sp75 reactivity. Both high-titered (1/16,000-1/32,000) and low-titered (1/1,000-1/4,000) IgM anti-sp75 reactive sera were found. IgM anti-sp75 antibodies persisted in the circulation over a long period in patients with fluctuating abnormal ALT levels. There was a striking association between detection of specific IgM anti-sp75 reactivity and the presence of HCV RNA in serum. Thus 11 of 15 (73%) sera containing HCV RNA also contained IgM anti-sp75 antibodies, while none of the HCV RNA-negative sera were IgM anti-sp75 reactive. Five of 11 patients without detectable levels of specific IgM anti-sp75 antibodies had their ALT levels returned to normal within 8 months to 3 years. Furthermore, a significant correlation was noted between the specific IgM anti-sp75 titers and the concentration of total plasma IgM, indicating that the immunological active region sp75 within the capsid of HCV has the capacity to induce an IgM secretion, which constitutes a substantial portion of the total plasma IgM, in patients with chronic HCV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis delta virus infection in Bombay.

From June 1985 to June 1989, sera from 425 cases of acute viral hepatitis were gathered from 2 hospitals in Bombay; 331 sera were positive for hepatitis B surface antigen and immunoglobulin M anti-hepatitis B core antigen, and the donors' disease was diagnosed as hepatitis B. Anti-hepatitis D virus was found in 124 of these sera, and hepatitis D antigen was present in 24 more, conclusively proving the presence of hepatitis delta infection in association with hepatitis B in Bombay. Among the 425 cases of hepatitis, 39 cases of fulminant hepatitis developed, of whom 31 died. Hepatitis B virus (HBV) was the apparent viral infection in 32 of the fulminant cases, and 20 (63%) of them also showed evidence of hepatitis D virus (HDV) infection, suggesting an aggravation of their clinical course due to concurrent HBV and HDV infections.

Characterization of a reduction-sensitive factor from human plasma responsible for apparent false activity in competitive assays for antibody to hepatitis B core antigen.

Addition of reducing agents to competitive assays for antibody to hepatitis B core antigen (anti-HBc) eliminates apparent false reactivity of specimens obtained from individuals with no prior history of hepatitis B virus (HBV) infection and without other serological markers of HBV infection. We have purified and characterized a reduction-sensitive factor (RSF) isolated from the plasma of several volunteer blood donors. Column fractions were assayed fro anti-HBc by using a highly sensitive chemiluminescence assay with a detection of 0.15 Paul Ehrlich Institut units per ml at 50% inhibition. Gel filtration on Sephacryl S-300 indicated that reductant-sensitive samples possessed anti-HBc activity that was associated with immunoglobulin M (IgM), whereas reductant-stable activity was associated with IgG. Gel filtration followed by metal chelate affinity chromatography resulted in a 55-fold purification and demonstrated that RSF activity copurifies with IgM. RSF was recovered from a recombinant hepatitis B core antigen matrix and shown to be an IgM species by immunoblot. In addition, RSF activity coeluted with IgM protein from anti-mu-chain Sepharose. Discrepancies between enzyme immunoassay and radioimmunoassay procedures for anti-HBc (Corzyme and Corab, respectively: Abbott Laboratories, North Chicago, Ill.) appear to be due to the relative sensitivity of the enzyme immunoassay for IgM anti-HBc (sevenfold greater than the radioimmunoassay using a specific panel). The biological basis for the occurrence of low levels of nonspecific IgM anti-HBc reactivity in individuals not previously exposed to HBV remains to be elucidated.

Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.

Mouse monoclonal antibody 5-21-3 recognizes a contiguous, conformation-dependent epitope and maps to a hydrophilic region in HIV-1 gp41.

Mouse monoclonal antibody 5-21-3 is mapped to an epitope within a hydrophilic region of HIV-1 gp41 between amino acids 642 and 665 (numbering by Meyers et al. based on HXB2 isolate). The epitope is formed from amino acids within the sequence IHSLIEESQNQQEKNEQELLELDK; however, antibody 5-21-3 is unable to recognize the epitope-forming sequence when it is presented to the antibody in the form of a short (642-665) synthetic polypeptide. The epitope apparently is partially formed when additional native sequence of varying length is added to the amino and/or carboxy ends of the epitope-forming sequence, and 5-21-3 binds these larger synthetic polypeptides to varying degrees depending on the position and length of the flanking sequences. The 5-21-3 epitope apparently is formed from contiguous amino acids which require a specific, conformation-dependent, secondary structure for proper epitope formation. Binding preferences exhibited by 5-21-3 toward synthetic polypeptides and recombinant proteins may reflect the conformational nature of the epitope in disrupted HIV which elicited formation of the monoclonal.

Diagnostic utility of a mouse monoclonal antibody (5-21-3) employed as a competitive probe in an immunoassay to detect antibody to HIV-1 gp41.

A mouse monoclonal antibody, designated 5-21-3, was raised against HIV-1 gp41 using detergent-disrupted virus as the immunogen. Antibody 5-21-3 was conjugated to horseradish peroxidase (HRP) and employed as a competitive probe against normal and HIV-1 antibody-positive sera in an immunoassay to detect the presence of antibody to HIV-1 gp41. The diagnostic utility of the competitive monoclonal immunoassay was assessed by correlation to a similar assay which employed HRP-labeled polyclonal IgG from a gp41-seropositive donor as the competitive probe. The monoclonal immunoassay was greater than 98% as sensitive and 99% as specific as the polyclonal immunoassay, regardless of the geographic source or disease state of the donor. The monoclonal immunoassay also was nearly as effective as the polyclonal immunoassay in detecting points of seroconversion in individuals enrolled in longitudinal studies. Of particular interest was the finding that the epitope recognized by monoclonal antibody 5-21-3 did not map to the well-characterized gp41 immunodominant region.

Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins.

We molecularly cloned the gag and env genes of the human immunodeficiency virus (HIV) and expressed fragments of these genes in Escherichia coli. Using the recombinant core and envelope proteins, we developed two competitive immunoassays (CIAs). Samples that recognized either the envelope or core proteins were considered positive for antibodies to HIV. This test system was comparable with western blot in detecting antibodies in patients with AIDS or AIDS-related complex that were repeatably reactive in the HIV screening test. All 360 individuals who were positive by western blot were positive by the CIA. A total of 844 samples repeatably reactive by an ELISA screening test were negative both by western blot and by the CIA; 48 samples positive by ELISA, but negative or indeterminate by western blot, were positive by the CIA. Alternate research procedures verified the positivity of these individuals. These data indicate that the CIA described here may be useful as an adjunct or alternative to the western blot.

Performance evaluation of the Abbott HTLV III EIA, a test for antibody to HTLV III in donor blood.

An enzyme immunoassay (EIA), designed to detect antibodies to human T-cell lymphotropic virus type III (HTLV III), was evaluated. The antibody test was found to be highly sensitive; serum from 221 of 223 (99.1%) patients with acquired immune deficiency syndrome (AIDS) was positive for antibodies to HTLV III. In addition, the antibody test was found to be highly specific; approximately 99.75% of 20,720 serum or plasma samples from blood donors were negative for antibody to HTLV III. In most cases, the Western Blot analysis agreed well with the EIA. Eighty-one of 82 (98.8%) EIA-positive samples from patients with AIDS were Western Blot positive. Of the EIA-positive blood donors, 21 of 36 (58%) were detected and confirmed by Western Blot analysis. A solid-phase competitive EIA also was evaluated as an alternate procedure. The preliminary results indicate that this immunoassay has a high degree of sensitivity and specificity and could serve as an alternate procedure to detect antibody to HTLV III.

Monoclonal antibodies to hepatitis A virus.

This paper describes the development of monoclonal antibodies generated against hepatitis A virus (HAV). Monoclonal antibodies (MCABs) from two murine hybridoma cell lines were found to bind to an epitope recognized in the sera of patients recovering from infection with HAV. Ascites fluids containing MCABs from one hybridoma (H1 C19) inhibited a maximum of 70% of the 125I-labeled polyclonal human anti-HAV from binding to HAV-antigen in a competitive radioimmunoassay, indicating that the MCAB recognizes a major epitope of HAV. Monoclonal anti-HAV that was coated onto polystyrene beads was as effective as polyclonal antibodies in capturing HAV antigen from extracts of human feces, marmoset liver, and cultured cells. Radiolabeled MCAB was used to screen sera for anti-HAV. A collection of 117 sera was tested for total anti-HAV by competitive radioimmunoassay utilizing either 125I-labeled human polyclonal or mouse monoclonal antibody. Thirty-four specimens were similarly reactive by both systems, while the remainder were negative. Likewise, 28 specimens were similarly positive for IgM anti-HAV, and 12 specimens were negative using each of the two labeled antibodies. The data show that anti-HAV induced during disease is directed against a common major epitope of HAV and that MC anti-HAV can be used effectively as a reagent to detect these antibodies.

Anti-hepatitis B core immunoglobulin M in the serologic evaluation of hepatitis B virus infection and simultaneous infection with type B, delta agent, and non-A, non-B viruses.

The clinical value of an enzyme-linked immunosorbent assay for the detection of immunoglobulin M (IgM) antibody to hepatitis B core antigen (anti-HBc IgM) was evaluated by testing serum samples from the following groups of patients: (a) 27 individuals who had been diagnosed as having acute hepatitis B virus (HBV) infection, (b) 29 hepatitis B surface antigen (HBsAg) carriers, (c) 6 subjects with acute non-B hepatitis, and (d) 10 HBsAg-negative but anti-HBc-positive subjects who were suspected of being index cases for the intimate transmission of HBV. Whereas 24 of the 27 individuals with presumed acute HBV infection exhibited anti-HBc IgM, only 2 of 29 HBsAg carriers were found to be positive. Hepatitis B surface antigen persisted during an 8-mo observation period in 3 anti-HBc IgM-negative subjects with acute HBsAg-positive hepatitis. Before anti-HBc IgM testing, it was considered that these cases had evolved to the HBsAg carrier state. However, the regular demonstration of anti-HBc IgM in acute type B hepatitis, as well as the failure to detect this antibody in the majority of HBsAg carriers, led to reclassification of these cases as probable instances of acute non-A, non-B or delta-agent hepatitis superimposed on the HBsAg carrier state. Through additional testing, the diagnosis of non-A, non-B (NANB) infection was confirmed in 2 of these cases, and delta-agent infection was identified in the third. None of the non-B hepatitis cases exhibited anti-HBc IgM. However, 5 of the 10 suspected type B index cases were anti-HBc IgM-positive, indicating that they were very recently infected and most likely had infected their cohabiting sexual partners. The results from this study indicate that testing for anti-HBc IgM may improve serodiagnostic accuracy when acute NANB and delta-agent hepatitis occur in previously unrecognized HBsAg carriers. Moreover, it may be a useful test in defining potential high risk sources of exposure to HBV.

Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc.

The time sequence, relative reactivity, and persistence of anti-HBc IgM were assessed in patients with HBsAg-positive viral hepatitis. A solid-phase immunoassay was developed using the IgM capture procedure with anti-mu-coated polystyrene beads. HBcAg was purified from serum Dane particles and used as a probe with 125I-labeled anti-HBc IgG. This immunoassay exhibited a pronounced prozoning phenomenon, and relative titers of sera differed widely depending upon the dilution of serum tested. When all sera were tested at 1:5,000 dilution, results were comparable in different patient groups. Anti-HBc IgM persisted at detectable levels for up to 2 years, and it was necessary to establish relative titers to discriminate current from remote infections. A cut-off assay value was established, and in 12 cases of acute hepatitis B virus (HBV) infection, antibody exceeded this value for about 6 months after onset of HBs antigenemia. A similar profile of anti-HBc IgM persistence was observed in seven patients who developed an HBsAg chronic carrier state. Long-term viral replication did not sustain elevated IgM class-specific antibody levels. The studies suggest that anti-HBc IgM analyses may be useful for differentiating recent and current HBV infections from remote infections, eliminating HBV as the agent for non-A, non-B hepatitis in asymptomatic HBsAg carriers, and detecting HBV as the etiologic agent during silent (HBsAg negative) infections.

Interpretation of various serological profiles of hepatitis B virus infection.

Serial serum specimens from 149 patients with clinically diagnosed hepatitis were tested for five hepatitis B serological markers: hepatitis B surface antigen and its antibody (anti-HBs); hepatitis B e-antigen and its antibody (anti-HBe); and antibody to hepatitis B core antigen (anti-HBc). The times of appearance, disappearance, and persistence of these markers were used to differentiate various serological profiles obtained from the study. Four distinctive profiles were found to be associated with acute hepatitis B followed by recovery, and three with chronic hepatitis. These serologic profiles were assessed as diagnostic and prognostic guides for clinical management of the disease.

Diagnosis of acute hepatitis A by HAVAB-M, a direct radioimmunoassay for IgM anti-HAV.

A three-step solid-phase radioimmunoassay, HAVAB-M, was developed for use in clinical labs as an aid to diagnosing hepatitis A. Polystyrene beads were coated with anti-human IgM (mu-chain specific) and were incubated successively with serum specimen, HAV, and anti-HAV 125I. HAVAB-M was used to assay sera from patients with hepatitis A and was found to have high sensitivity for the IgM antibody to HAV. The antibody was detectable within a few days of onset of symptoms of hepatitis, and it reached maximum concentrations within one to three weeks. The test was designed so that most patients' sera would become negative approximately three months after onset. HAVAB-M was shown to be specific for IgM antibody, with virtually no detection of IgG anti-HAV. The test showed no interference fro rheumatoid factor and no cross-reactivity with sera from patients with hepatitis B or other infectious diseases.