Not the presence but the amount of clonal DNA detectable in remission of acute myeloid leukemia is predictive for relapse.

OBJECTIVES: The persistence of clonal cells after chemotherapy, or a re-emerging of clonal cells in remission (CR) or at relapse in patients with acute myeloid leukemia (AML) was studied to assess the prognostic significance of the amount of clonal DNA in predicting the clinical outcome. METHODS: Clonal rearrangements in the gene sequences of retinoic acid receptor (RAR) alpha, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T-cell receptor (TcR) beta, myeloid lymphoid leukemia or cytokines (GM-CSF, G-CSF, IL-3) detected in bone marrow samples from 37 patients with primary AML (pAML) or secondary AML (sAML) were investigated. A relative increase or decrease of clonal DNA in the course of AML was evaluated by comparing the optical densities of DNA bands of the rearranged genes and the total amount of DNA. RESULTS: High amounts of clonal DNA were detectable at diagnosis, during persisting disease and at relapse (Ø 39%, 35%, or 38% of total DNA, respectively), compared to 20% in complete remission (CR). Amounts of clonal DNA (except for Ig-JH gene rearrangements) were of prognostic significance at diagnosis, patients with less than 33% clonal DNA were characterized by significantly longer relapse-free survival times (all cases: p = 0.01; pAML: p = 0.002). Patients in CR exhibiting less than 5% (all cases) or 15% (pAML) clonal DNA showed longer relapse-free survival times (p = 0.08 or p = 0.03, respectively). Vice versa, significantly higher amounts of clonal DNA (all cases 51% vs. pAML 54%) could be detected in cases studied at diagnosis who relapsed in the following 5 months (all cases p = 0.01) or 14 months (pAML p = 0.007). Significantly higher amounts of clonal DNA (33%) could be detected in cases studied in CR who relapsed in the following 4 months (all cases p = 0.002 or pAML p = 0.006, respectively). Moreover, we could prove disease progression on a cellular level months before the clinical onset of sAML after a period of MDS. CONCLUSIONS: Clonal, gene-rearranged DNA is regularly detectable at diagnosis and during persisting AML, in CR and at relapse. However, the presence, rather than the amount of clonal DNA detectable in CR is predictive for relapse. These data might indicate the significance of gene rearrangement analyses in the course of AML to identify cases with a high risk of relapse, independently from the karyotype.

Gene rearrangements in bone marrow cells of patients with acute myelogenous leukemia.

At diagnosis, clonal gene rearrangement probes [retinoic acid receptor (RAR)-alpha, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T cell receptor (TcR)-beta, myeloid lymphoid leukemia (MLL) or cytokine genes (GM-CSF, G-CSF, IL-3)] were detected in bone marrow samples from 71 of 153 patients with acute myelogenous leukemia (AML) (46%): in 41 patients with primary AML (pAML) (58%) and in 30 patients with secondary AML (42%). In all cases with promyelocytic leukemia (AML-M3) RAR-alpha gene rearrangements were detected (n = 9). Gene rearrangements in the Ig-JH or the TcR-beta or GM-CSF or IL-3 or MLL gene were detected in 12, 10, 16 and 12% of the cases, respectively, whereas only few cases showed gene rearrangements in the M-bcr (6%) or G-CSF gene (3%). Survival of pAML patients with TcR-beta gene rearrangements was longer and survival of pAML patients with IL-3 or GM-CSF gene rearrangement was shorter than in patients without those rearrangements. No worse survival outcome was seen in patients with rearrangements in the MLL, Ig-JH or M-bcr gene. In remission of AML (CR), clonal gene rearrangements were detected in 23 of 48 cases (48%) if samples were taken once in CR, in 23 of 26 cases (88%) if samples were taken twice in CR and in 23 of 23 cases (100%) if samples were studied three times in CR. All cases with gene rearrangements at diagnosis showed the same kind of rearrangement at relapse of the disease (n = 12). Our data show that (1) populations with clonal gene rearrangements can be regularly detected at diagnosis, in CR and at relapse of AML. (2) Certain gene rearrangements that are detectable at diagnosis have a prognostic significance for the patients' outcome. Our results point out the significance of gene rearrangement analyses at diagnosis of AML in order to identify 'poor risk' patients - independently of the karyotype. Moreover, the persistence of clonal cells in the further course of AML can be studied by gene rearrangement analysis.

The formation of ceramide from sphingomyelin is associated with cellular apoptosis.

The apoptotic response of the immature B-cell to the cross-linking of surface IgM receptors provides a good model for cell death and we show in WEHI-231 B-cells that the time course of apoptosis corresponds to the increased formation of ceramide, as measured either by mass (using the diacylglycerol kinase method) or radiolabelling with [3H]palmitate. Inhibitors of sphingosine biosynthesis have no effect on cell death induced by anti-IgM in WEHI-231 but inhibitors of ceramidase accelerate apoptosis, suggesting that activation of sphingomyelinase is the key event in apoptosis. We have demonstrated this by in vitro assay of neutral sphingomyelinase. Apoptosis is also important in normal brain development and neuronal survival is dependent upon phosphatidylinositol 3-kinase (PI3-kinase) activation by growth factors (insulin, nerve growth factor etc.). Withdrawal of these growth factors or inhibition of PI3-kinase with wortmannin or LY294002 activated the pro-apoptotic CPP32 (Yama/Apopain/caspase 3, EC 3.4.22), activated neutral sphingomyelinase and increased ceramide formation in an immortalized dorsal root ganglion cell line F-11. Protection against apoptosis can be achieved by overexpression of the bc12 family of proteins or addition of drugs which elevate cAMP levels. cAMP protects against apoptosis induced by either wortmannin or staurosporine. The specificity for cAMP was confirmed by showing protection with the specific agonist (Sp)cAMPS and increased killing with the antagonist (Rp)cAMPS. However, cAMP did not protect against ceramide killing, suggesting that there are at least two major pathways of apoptosis in neuronal cells.

Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL.

Prolonged (>24 h) exposure to anti-IgM (an antigen surrogate that induces membrane cross-linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by 32P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [3H]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure times to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elicit increased ceramide formation. After 8 h, apoptosis occurs concomitantly with ceramide formation over the next 40 h. Further, we showed that exogenous ceramide mimicked anti-IgM-induced apoptosis and that apoptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased both ceramide formation and apoptosis and accelerated apoptosis induced by anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resistant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis induced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to be the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in neutral sphingomyelinase activity observed in wild type cells. We have shown previously that transfection of WEHI cells with bcl-xL conferred resistance to anti-IgM-induced apoptosis, whereas transfection with bcl-2 did not (Gottschalk, A., Boise, L., Thompson, C., and Quintans, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7350-7354). In this study, these bcl-xL transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C2-ceramide) or N-hexanoylsphingosine (C6-ceramide). However, when challenged with anti-IgM the bcl-xL transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-xL and that it is a major determinant of B-cell death.

Staurosporine induces programmed cell death in embryonic neurons and activation of the ceramide pathway.

We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1-1.0 microM) and observed the meporphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N-Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [3H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 microM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.

Programmed cell death in neurotumour cells involves the generation of ceramide.

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 microM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.

Natural history of HIV-1 cell-free viremia.

OBJECTIVE: To characterize the natural history of viremia with human immunodeficiency virus type 1 (HIV-1) and its association with disease progression from infection to acquired immunodeficiency syndrome (AIDS). DESIGN: Prospective cohort study. Annual specimens were tested for quantitative virion-associated HIV-1 RNA, p24 antigen, and CD4+ lymphocyte levels. PARTICIPANTS: A total of 42 homosexual men who seroconverted to HIV-1 between 1982 and 1985. MAIN OUTCOME MEASURES: Trends over time in serum HIV-1 RNA level, correlations between serum HIV-1 RNA and other markers, and prediction of AIDS using these markers. RESULTS: HIV-1 RNA levels were stable over time, increasing by 10-fold or more in only six (14%) of the 42 subjects during 3 to 11 years of follow-up. Mean HIV-1 RNA levels were 10(3.8) copies/mL if AIDS occurred in less than 4 years, 10(3.07) copies/mL if AIDS developed within 4 through 9 years, and 10(2.27) copies/mL if AIDS did not develop within 6 through 11 years. In both univariate and multivariate models, initial and subsequent HIV-1 RNA levels, p24 antigenemia, and percentage of CD4+ lymphocytes were independently predictive of AIDS. CONCLUSIONS: The stability of virion-associated HIV-1 RNA levels suggests that an equilibrium between HIV-1 replication rate and efficacy of immunologic response is established shortly after infection and persists throughout the asymptomatic period of the disease. Thus, defective immunologic control of HIV-1 infection may be as important as the viral replication rate for determining AIDS-free survival. Because individual steady-state levels of viremia were established soon after infection, HIV-1 RNA levels may be useful markers for predicting clinical outcome.

Detection of p24 antigen with and without immune complex dissociation for longitudinal monitoring of human immunodeficiency virus type 1 infection.

Sequential specimens obtained from 87 multicenter AIDS cohort study participants were tested by three p24 antigen tests. They included a polyclonal enzyme immunoassay (EIA), a monoclonal EIA, and a monoclonal EIA after immune complex dissociation (ICD) of specimens. Subjects were grouped into two categories defined by real-time testing with the polyclonal EIA: 39 had become positive for p24 antigen (antigen converters) during follow-up, and 48 had progressed to AIDS without detectable antigenemia. Twenty-four (61%) antigen converters were positive by ICD-monoclonal EIA about 1 year earlier than by monoclonal EIA. In contrast, only 12 (25%) patients who progressed to AIDS without detectable antigenemia became positive by ICD-p24 EIA before developing AIDS. Thus, the main benefit of ICD treatment may be to detect p24 antigenemia approximately 1 year before the regular assay rather than to identify additional antigenemic people. Quantitative plasma RNA levels were also determined in longitudinal samples from 20 antigen converters and 7 men who developed AIDS without antigenemia. Although mean human immunodeficiency virus type 1 RNA levels were higher in antigen-positive than in antigen-negative samples (P = 0.002), more than half (11 of 20) of the antigen converters had no measurable change in human immunodeficiency virus type 1 RNA associated with change to antigen positivity.

Circulating gangliosides of breast-cancer patients.

Gangliosides were isolated from the sera of recently diagnosed breast-cancer patients and from individuals who were apparently free of disease. Quantificative and qualitative analyses were carried out by 2-dimensional high-performance thin-layer chromatography and gas chromatography. The locations of isolated gangliosides on thin-layer chromatograms were determined by visualization with resorcinol, and each spot was quantified by digital image densitometry. The ganglioside profiles of cancer patients were compared to those of the control group, revealing a significant increase in total lipid-bound sialic acid and a specific increase in polysialogangliosides in the patients with breast cancer. Furthermore, an increase was noted in the ratio of gangliosides of the b-series biosynthetic pathway over those of the a-series in the cancer sera, as compared to the controls. Gas chromatographic analysis of the peracetylated methanolysis mixtures derived from the total ganglioside fraction of cancer patients supported the HPTLC data, with an increase in total sialic acid, galactose, and sphingosine residues. No unusual gangliosides were found in the mixture from breast-cancer patients.

The mediating role of anxiety in self-reported somatic complaints of depressed adolescents.

This study examined the mediating role of anxiety in the self-reports of somatic complaints in 96 depressed adolescent inpatients. Sixty-four subjects with major depressive episodes and comorbid anxiety disorders (MDE-A) determined from the Diagnostic Interview for Children and Adolescents--Revised (DICA-R) reported significantly more somatic complaints than 32 adolescents having major depressive episodes without comorbid anxiety (MDE). An analysis of covariance demonstrated that, with anxiety symptoms controlled, MDE and MDE-A groups did not differ significantly in somatic complaints. A hierarchical multiple-regression analysis revealed that, with demographic and anxiety symptoms controlled, depressive symptoms did not contribute to the explanation or prediction of somatic complaints. The results suggest that anxious, but not depressive symptoms, are independently associated with somatic complaints. The results are discussed in light of new affective models of psychopathology.

Gender and the comparison of self and observer ratings of anxiety and depression in adolescents.

OBJECTIVE: This study assessed the influence of gender on the comparability of self and observer ratings of anxiety and depression in adolescents. METHOD: Subjects were 75 inpatient adolescents who were administered structured interviews of the revised Hamilton Rating Scales for Depression (HRSD-R) and Anxiety (HARS-R) and read the Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI). RESULTS: All measures demonstrated adequate internal consistency and validity. The correlation between the BDI and HRSD-R was significantly higher for females than males; of 11 symptoms that overlap on the BDI and HRSD-R, observers significantly agreed with males and females in their perceptions of 5 and 11 depressive symptoms, respectively. The correlation between the BAI and HARS-R did not differ significantly for males and females. CONCLUSIONS: Results suggest that self-reports of anxiety symptoms are a valid, cost-effective alternative to anxiety observer ratings for boys and girls' self-reports of depression are comparable to depression ratings by observers. There is the need to collect self-report information from adolescent boys because they may not communicate subjective symptoms of depression, e.g., guilt, to observers.

Microscale analysis of glycosphingolipids by methanolysis, peracetylation, and gas chromatography.

A method for the analysis of pure samples of individual glycosphingolipids by microscale methanolysis, peracetylation, and gas chromatography is described. Solvolysis of glycosphingolipids in dry methanolic HCl and peracetylation were conducted in a single 4.5-cm sealed capillary tube (2 mm i.d.), after which the products were directly injected into a gas chromatograph. Total-component analysis (i.e., analysis of the sugar, fatty acid, and sphingosine moieties) was possible after a 45-min chromatographic run. Time-course studies of the acid-catalyzed methanolysis of Gal beta 1-4GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer ganglioside at 80, 110, and 150 degrees C showed that methanolysis was complete after 2 h at 110 degrees C. Rates of methanolysis of individual components were compared and the release of the fatty acid moiety from the long-chain base was shown to be the slowest reaction. The methanolysis of all glycosidic bonds were complete in 0.5 h. Peracetylated methanolysis products were very stable over time and provided for good gas chromatographic detection of subnanomolar amounts of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphingoid base components. Recoveries of fucose and N-acetylglucosamine were determined with reference samples of Fuc alpha 1-2Lac and lacto-N-fucosylpentaose II. Applications of the method are presented for the component analysis of a gift mixture of NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-1Cer ganglioside and NeuAc alpha 2-6Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer ganglioside and analysis of NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer isolated from human plasma.

Choosing alternative therapy: an exploratory study of sociodemographic characteristics and motives of patients resident in Sydney.

Disenchantment with allopathic medicine has coincided with an upsurge in recent years in Australians consulting alternative therapists about their health care needs. Two major government studies have provided valuable independent evidence about the sociodemographic characteristics of users of alternative therapies and about their attitudes to health and medical care. However, the focus of these inquiries was predominantly on the users of chiropractic and osteopathy, just two of the modalities which make up this diverse field. To investigate, among other things, who consults alternative practitioners, how they come to choose a particular therapist, and whether, and on what basis, they have abandoned cosmopolitan medical care or use a combination of alternative and allopathic medicine, we conducted a cross-sectional survey of 289 patients of eight Sydney practices providing a range of alternative modalities. Findings indicate that far from being representative of the Australian community, the majority of our sample population came from a very select group, with a narrow range of socioeconomic backgrounds. The health risk behaviour of those surveyed was also significantly different from that exhibited by the population in general.

RETROCELL HIV-1 passive hemagglutination assay for HIV-1 antibody screening.

A passive hemagglutination assay (PHA) for serum human immunodeficiency virus type 1 (HIV-1) antibody screening was developed using aldehyde-stabilized human erythrocytes coated with purified HIV-1 antigens. The assay is simple to perform, and the components are inexpensive. In preliminary testing of a panel of 490 confirmed HIV-1 seropositive specimens, all tested positive by the HIV-1 PHA assay. By testing seroconversion specimens and dilution panels, the assay demonstrated a sensitivity equivalent to a recombinant protein-based HIV-1 enzyme immunoassay (EIA). In-house evaluation of 2,557 HIV-1 prescreened specimens from South African blood bank donors revealed initial and repeat reactive rates of 0.2 and 0.04%, respectively. Field testing of the HIV-1 antibody PHA at two sites gave performance of 100% sensitivity (400 seroconfirmed samples) and 0.55% initial/0.28% repeat reactive rates in the testing of 3,983 negative samples. The performance, low cost, and ease of use make the HIV-1 antibody PHA test a prime candidate for HIV-1 antibody screening in regions of the world where more sophisticated technology is inappropriate.

A caste and outcaste system in medicine.

The development and establishment of caste-like divisions among health-care practitioners is examined with particular attention being paid to chiropracty. Reference is made to recent studies and changes in Australasia. Comparisons are drawn between changes occurring in medicine and the persistence of hierarchical arrangements which exclude alternative therapies, such as chiropracty.