High-dose cyclophosphamide in severe aplastic anaemia: a randomised trial.

BACKGROUND: High-dose cyclophosphamide has been proposed as an alternative immunosuppressive agent for treatment of severe aplastic anaemia, with a response rate similar to that with regimens containing antithymocyte globulin (ATG) but neither relapse nor clonal haematological complications. We undertook a phase III, prospective, randomised trial to compare response rates to immunosuppression with either high-dose cyclophosphamide plus cyclosporin or conventional immunosuppression with ATG plus cyclosporin in previously untreated patients. METHODS: Between June, 1997, and March, 2000, 31 patients were enrolled. 15 were assigned cyclophosphamide (1 h intravenous infusion of 50 mg/kg daily for 4 days) and 16 were assigned ATG (40 mg/kg daily for 4 days); both groups received cyclosporin, initially at 12 mg/kg daily with adjustment to maintain concentrations at 200-400 microg/L, for 6 months. The primary endpoint was haematological response (no longer meeting criteria for severe aplastic anaemia). The trial was terminated prematurely after three early deaths in the cyclophosphamide group. Analyses were by intention to treat. FINDINGS: Median follow-up was 21.9 months (range 1-33). There was excess morbidity in the cyclophosphamide group (invasive fungal infections, four cyclophosphamide vs no ATG patients; p=0.043) as well as excess early mortality (three deaths within the first 3 months cyclophosphamide vs no ATG patients; p=0.101). There was no significant difference at 6 months after treatment in the overall response rates among evaluable patients (six of 13 [46%] cyclophosphamide vs nine of 12 [75%] ATG). INTERPRETATION: A longer period of observation will be necessary to assess the secondary endpoints of relapse and late clonal complications as well as disease-free and overall survival. However, cyclophosphamide seems a dangerous choice for treatment of this disorder, given the good results achievable with standard therapy.

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Cyclophosphamide and other new agents for the treatment of severe aplastic anemia.

Severe aplastic anemia (SAA) has a poor prognosis in the absence of treatment. Current accepted therapeutic strategies include allogeneic stem-cell transplantation and immunosuppression, both resulting in long-term survival in the majority of patients. Although human leukocyte antigen (HLA)-matched sibling stem-cell transplantation is highly effective, the 25% probability of finding a suitable sibling donor within a family renders this approach available to only a minority of patients. Transplantation using HLA-matched, unrelated donors carries a high risk of treatment failure along with considerable toxicity. While combined immunosuppression with both antithymocyte globulin (ATG) and cyclosporine A (CSA) produces hematologic improvement in most patients, relapse is common. Late evolution of aplastic anemia to other serious hematologic disorders, including paroxysmal nocturnal hemoglobinuria (PNH), myelodysplasia, and acute leukemia, is also a significant problem following treatment with ATG/CSA. Recently, results of immunosuppression in SAA with another potent immunosuppressive agent, cyclophosphamide, were reported in a small number of patients. The overall response rate was similar to that seen with ATG/CSA, but relapse and late clonal disease were not observed during a long period of follow-up. A larger randomized trial comparing sustained hematologic response rates to either conventional immunosuppression with ATG/CSA or high-dose cyclophosphamide and CSA is now underway; secondary end points include response duration, event-free survival, and overall survival. Additionally, a number of protocols designed to test the efficacy of alternative immunosuppressive or immunomodulatory agents are being developed.

Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem-cell disorder in which the affected cells are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins. Paroxysmal nocturnal hemoglobinuria is frequently associated with aplastic anemia, although the basis of this relation is unknown. OBJECTIVE: To assess the PNH status of patients with diverse marrow failure syndromes. DESIGN: Correlation of cytofluorometric data with clinical features. SETTING: Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland. PATIENTS: 115 patients with aplastic anemia, 39 patients with myelodysplasia, 28 patients who had recently undergone bone marrow transplantation, 18 patients with cancer that was treated with chemotherapy, 13 patients with large granular lymphocytosis, 20 controls who had received renal allografts, and 21 healthy participants. INTERVENTION: Patients with aplastic anemia, myelodysplasia, or renal allografts received antithymocyte globulin. MEASUREMENTS: Flow cytometry was used to assess expression of GPI-anchored proteins on granulocytes. RESULTS: Evidence of PNH was found in 25 of 115 (22%) patients with aplastic anemia. No patient with normal GPI-anchored protein expression at presentation developed PNH after therapy (n = 16). Nine of 39 (23%) patients with myelodysplasia had GPI-anchored protein-deficient cells. Abnormal cells were not detected in patients with constitutional or other forms of bone marrow failure or in renal allograft recipients who had received antithymocyte globulin. Aplastic anemia is known to respond to immunosuppressive therapy; in myelodysplasia, the presence of a PNH population was strongly correlated with hematologic improvement after administration of antithymocyte globulin (P = 0.0015). CONCLUSIONS: Flow cytometric analysis is superior to the Ham test and permits concomitant diagnosis of PNH in about 20% of patients with myelodysplasia (a rate similar to that seen in patients with aplastic anemia). The presence of GPI-anchored protein-deficient cells in myelodysplasia predicts responsiveness to immunosuppressive therapy. Early emergence of GPI-anchored protein-deficient hematopoiesis in a patient with marrow failure may point to an underlying immune pathogenesis.

Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-gamma (IFN-gamma) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-gamma with those that regulate chemokine receptor expression.

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures.

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38-) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Regulation of a graft-versus-leukemia effect by major histocompatibility complex class II molecules on leukemia cells: HLA-DR1 expression renders K562 cell tumors resistant to adoptively transferred lymphocytes in severe combined immunodeficiency mice/nonobese diabetic mice.

To understand the role of key molecules in determining the strength and nature of allogeneic T-cell response to leukemia, we transfected HLA-DR1 into the major histocompatibility complex (MHC)-deficient, natural killer (NK)-cell sensitive K562 leukemia cell line. Untransfected K562 cells stimulated NK proliferation in vitro and formed subcutaneous tumors in severe combined immunodeficiency/non-obese diabetic (SCID/NOD) mice. Tumor growth was inhibited by adoptive intravenous transfer of fresh unprimed peripheral blood mononuclear cells (PBMC). In contrast, HLA-DR1 transfected cells stimulated CD4(+) T cells, but not NK-cell proliferation in vitro and formed tumors resistant to fresh PBMC in SCID/NOD mice. Tumors not expressing MHC were infiltrated with CD16(+)CD56(+) lymphocytes whereas nonregressing HLA-DR1 expressing tumors showed only a scanty infiltration with both T-cell and NK-cell subsets. The results indicate that MHC class II expression by leukemia cells can determine the effector cell type that it engages. In vivo MHC class II expression rendered K562 cell tumors resistant to NK-cell mediated antitumor reactivity.

Toluene-induced hearing loss among rotogravure printing workers.

OBJECTIVES: This study explored the effects of occupational exposure to solvents and noise on the hearing of rotogravure printing workers from São Paulo, Brazil. METHODS: The study group comprised 124 workers exposed to various levels of noise and an organic solvent mixture of toluene, ethyl acetate, and ethanol. Data on work history, psychosocial aspects of the job, medical history, present health, stress, occupational and nonoccupational exposures to noise or chemicals, and life-style factors were collected through an interview. The participants underwent pure-tone audiometry and immittance audiometry testing. Their exposures to noise and solvents were assessed. RESULTS: Forty-nine percent of the workers had hearing loss. From the numerous variables that were analyzed for their contribution to the development of hearing loss (age, tenure, noise dose, solvent concentrations in air, biological marker for toluene, job category, work and medical history items, smoking, alcohol consumption, work perception scores, nonoccupational exposures), age and hippuric acid (the biologic marker for toluene in urine) were the only variables that met the significance level criterion in the final multiple logistic regression model. The odds ratio estimates for hearing loss were 1.07 times greater for each increment of 1 year of age [95% confidence interval (95% CI) 1.03-1.11] and 1.76 times greater for each gram of hippuric acid per gram of creatinine (95% CI 1.00-2.98). CONCLUSIONS: The findings suggest that exposure to toluene has a toxic effect on the auditory system. Further research is needed on the mechanisms underlying the effects of toluene and on the adequacy of current recommended exposure limits.

Paroxysmal nocturnal hemoglobinuria: new insights from murine Pig-a-deficient hematopoiesis.

A large fraction of the hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are deficient in membrane expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Current evidence suggests that this deficiency is sufficient to account for the hemolytic and thrombotic manifestations of this disease but not for its frequent association with aplastic anemia, an autoimmune disorder in which the patients' own hematopoietic progenitor cells are the target. Mutations in X-linked gene PIG-A, encoding one of several enzymes required for the biosynthesis of the glycophosphatidylinositol anchor, have been found in all PNH patients studied to date. Recent experiments with murine Pig-a knock-out embryonic stem cells show that although embryogenesis is critically dependent on normal GPI-AP expression, Pig-a-deficient cells can undergo apparently normal hematopoietic differentiation if they develop in a GPI-AP-replete environment. Thus, in an in vitro mouse model of PNH, Pig-a mutations confer no gross proliferative or differentiative advantage or disadvantage, suggesting an unidentified process selecting for these mutations in the bone marrow of patients with the PNH-aplastic anemia syndrome. The rescue of hematopoiesis observed in chimeric cultures of knock-out and normal cells was accompanied by intercellular transfer of GPI-AP, suggesting exciting new possibilities for future therapeutic manipulations in PNH patients.

Hearing loss from combined exposures among petroleum refinery workers.

Workers from a refinery (n = 438) were interviewed, had their hearing tested and had their exposures to noise and solvents assessed. Measurements suggested that most exposures to noise and solvents were within exposure limits recommended by international agencies; however, the prevalence for hearing loss within the exposed groups ranged from 42 to 50%, significantly exceeding the 15-30% prevalence observed for unexposed groups. The adjusted odds ratio estimates for hearing loss were 2.4 times greater for groups from aromatics and paraffins (95% CI 1.0-5.7), 3 times greater for the maintenance group (95% CI 1.3-6.9) and 1.8 times greater for the group from shipping (95% CI 0.6-4.9), when compared to unexposed workers from the warehouse and health clinic. The results of acoustic reflex decay tests suggest a retrocochlear or central auditory pathway involvement in the losses observed in certain job categories. These findings indicate that factors in addition to noise ought to be considered when investigating and preventing occupational hearing loss.

A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.

We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.

Auditory and vestibular functions after single or combined exposure to toluene: a review.

Toluene is a widely used organic solvent, heavily employed in many manufacturing industries. Recently, evidence has begun to accumulate on the deleterious effect of toluene exposure has on the auditory and vestibular systems. Although little published information exists regarding these effects, the reported findings indicate a need for further investigation. The results of such investigations may dramatically affect occupational hearing conservation practices and legislation. Both human and animal studies will be summarized in discussing the effects of toluene alone or in combination with noise or other chemicals. Gaps in scientific knowledge are highlighted to assist future research.

Occupational exposure to noise and ototoxic organic solvents.

The objectives of this study were to review the literature on the effects of occupational exposure to organic solvents on the auditory system and to identify work settings in which exposure to these agents and to noise might occur. The criteria for selecting the chemicals were (a) evidence available that indicated that the chemicals may affect the auditory system and enhance noise effects, and (b) the ubiquity of their use. References to ototoxicity were noted for three proven neurotoxicants, i.e., carbon disulfide, toluene, and trichloroethylene, and for two probable human neurotoxicants--styrene and xylene. The percentages of workers (estimated by NIOSH National Occupational Exposure Survey) exposed to these solvents in each economic sector are shown. Work settings are identified where multiple exposures occur to solvents and noise. The need for future research is discussed.

Effects of occupational exposure to organic solvents and noise on hearing.

This study explored the effects of occupational exposure to solvents and noise on hearing. Interviews and hearing tests were conducted for printing and paint manufacturing workers. The experimental groups included unexposed (N = 50) workers and workers exposed to noise (N = 50), noise and toluene (N = 51), or an organic solvent mixture (N = 39). The risk of hearing loss was greater for the exposed groups than for the unexposed group. The adjusted relative risk estimates were four times greater [95% confidence interval (95% CI) 1.4-12.2] for the noise group, 11 times greater (95% CI 4.1-28.9) for the noise and toluene group, and five times greater (95% CI 1.4-17.5) for the solvent-mixture group. The findings suggest that exposure to the studied solvents had a toxic effect on the auditory system and that an interaction between noise and toluene took place. The audiological results of the noise and toluene group suggest a central auditory pathway involvement in the hearing losses observed.

Hearing loss in the chinchilla from impact and continuous noise exposure.

The relative hazard posed to the peripheral auditory system by impact/impulse and continuous noise of the same power spectrum was determined. Impact noise was generated by striking a nail with a hammer and was digitally recorded. The acoustical power spectrum of the impact was determined and pink noise was filtered to produce a continuous noise stimulus with the same acoustic power spectrum. Pre-exposure auditory evoked response (AER) thresholds were obtained at 1, 2, 4, and 8 kHz on 16 adult chinchillas. The pool of animals was divided into two equal groups based upon pre-exposure AER thresholds. One group was exposed to impact noise and the other group to the filtered pink noise. Exposures were 4 h/day for 5 days. Thirty days following the exposure, auditory evoked response thresholds were remeasured. Changes in auditory sensitivity were determined by subtracting the pre-exposure thresholds from the post-exposure thresholds. Hearing threshold shifts of the impact noise group were significantly greater (p less than 0.0001) than the hearing threshold shifts of the continuous noise group. These data indicate a need to more closely examine the parameters and effects of impact noise. There may be a need to develop expanded damage-risk criteria for occupational exposure to impulse/impact noise.

Quantitative evaluation of scanning electron microscopy-examined ciliary morphological changes in control and noise exposed guinea pig cochleas.

Many investigations of noise-induced hearing loss have demonstrated a poor correlation between hearing threshold and hair cell loss. One reason for this is that more subtle changes in the hair cell, such as detailed morphological changes of stereocilia, have not been evaluated. However, examining such changes increases the problem of distinguishing experimental pathological changes from artefacts. Preparation of the specimen for scanning electron microscopy (SEM) may result in too many artefacts for an adequate quantification of defects due to noise exposure. One problem with some earlier studies seems to be lack of controls and/or statistical analysis for the purpose of eliminating the influence of artefacts and spontaneous degeneration. The aim of this study was to compare unexposed and noise-exposed cochleas examined with SEM in order to determine if subtle changes due to noise could be distinguished from preparation artefacts and from spontaneous deterioration. Ten different types of hair cell changes were found in exposed and control animals. By means of using controls for statistical comparison with noise-exposed animals two cell damage categories--hair cell loss and missing stereocilia--were found to be produced by exposure to noise.

An alternative pathway of induction of lymphokine production by T lymphocyte clones.

We have previously described a variant murine CTL clone that in contrast to all other clones tested, exhibited a novel capacity to produce IFN-gamma in response to IL-2. This alternative pathway of IFN-gamma induction differed from the conventional TCR complex-mediated pathway in that it was independent of elevated intracellular Ca2+ and insensitive to cyclosporine A. We report here the presence of an analogous pathway in the majority of T lymphocyte clones tested, when these clones are stimulated with IL-2 in the presence of syngeneic or third-party splenocytes. The accessory function of splenocytes in this alternative pathway is mediated by the MAC-1+ subpopulation and apparently involves cell-cell contact. However, the structure with which the MAC-1 antibody reacts probably is not involved directly. No involvement of Ag or the TCR for Ag could be demonstrated in this alternative pathway of lymphokine induction. The array of lymphokines induced by this alternative pathway is only a subset of those induced by antigenic stimulation. Finally, as with the previously described variant clone, IL-2-mediated induction of IFN-gamma production by the normal T lymphocyte clones is independent of normal extracellular Ca2+ levels and insensitive to cyclosporine A. Thus, this alternative pathway of lymphokine induction apparently constitutes a distinct signaling pathway in cloned T lymphocytes.

Production of IL-2 and IFN by TH2 clones.

We have studied the production of IL-2, IL-4 and IFN-gamma by a panel of CD4+ clones produced in our laboratory. The clones were classified as TH1 and TH2 because of their ability to secrete IL-2 or IL-4, respectively, following stimulation with APC + Ag and by their characteristic proliferative responses to exogenous IL-2 or IL-4. Some of the TH2 clones, all of which happened to be autoreactive, produced IL-2 and one of these, as well as one antigen-reactive TH2 clone, also secreted IFN-gamma following stimulation with immobilized anti-CD3 mAb. IL-2 production by TH2 cells required higher concentrations of anti-CD3 mAb than IL-4 production. Thus, the TH2 clones seem to be heterogeneous. We designate the IL-2/IL-4 secretors as TH2B and those making IL-4 as TH2A clones.