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Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by hemolytic anemia, hemoglobinuria, bone marrow failure, and hypercoagulability. Thrombosis is the leading cause of mortality and occurs in one-half of PNH patients, with the hepatic veins being the most common site. Patients with hepatic vein thrombosis (Budd-Chiari syndrome) can present with abdominal pain, hepatomegaly, jaundice, and ascites. Prognosis is poor for these patients; death may occur from liver failure, vessel rupture, intestinal ischemia, infarction, necrosis, or sepsis. The authors report three consecutive cases of successful treatment with catheter-directed thrombolysis and thrombectomy directly in the hepatic veins in patients with PNH who developed acute hepatic vein thrombosis. This treatment represents a potential bridge toward more curative therapies such as allogeneic bone marrow transplant.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon intravascular hemolytic anemia that results from the clonal expansion of hematopoietic stem cells harboring somatic mutations in an X-linked gene, termed PIG-A. PIG-A mutations block glycosylphosphatidylinositol (GPI) anchor biosynthesis, resulting in a deficiency or absence of all GPI-anchored proteins on the cell surface. CD55 and CD59 are GPI-anchored complement regulatory proteins. Their absence on PNH red cells is responsible for the complement-mediated intravascular hemolysis. Intravascular hemolysis leads to release of free hemoglobin, which contributes to many of the clinical manifestations of PNH including fatigue, pain, esophageal spasm, erectile dysfunction and possibly thrombosis. Interestingly, rare PIG-A mutations can be found in virtually all healthy control subjects, leading to speculation that PIG-A mutations in hematopoietic stem cells are common benign events. However, negative selection of PIG-A mutant colony-forming cells with proaerolysin, a toxin that targets GPI-anchored proteins, reveals that most of these mutations are not derived from stem cells. Recently, a humanized monoclonal antibody directed against the terminal complement protein C5 has been shown to reduce hemolysis and greatly improve symptoms and quality of life for PNH patients.

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.

An unusual case of aplastic anemia presenting in association with lymphoplasmacytic lymphoma and paroxysmal nocturnal hemoglobinuria is discussed. An insult to the hematological stem cell compartment may result in multiple pathological entities, potentially influencing our approach to the treatment of hematological clonal disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.

Purpose of Review: Autoimmune neurologic diseases are being increasingly recognized, and treated with conventional immunosuppressive agents. Patients with 'refractory' conditions have been treated with high-dose therapy, with or without autologous stem cell transplants. This paper reviews the rationale, methods, and recent results of high-dose therapy and the questions that it raises., Recent Findings: High-dose therapy has been used in progressive multiple sclerosis and in myasthenia gravis and autoimmune neuropathies that are refractory to conventional immunotherapy. A variety of methods of immune ablation have been used; most require hematopoietic 'rescue' with stem cell transplantation. High-dose cyclophosphamide alone is immunoablative but not myeloablative, permitting the patient's endogenous stem cells to repopulate the hematopoietic/immune systems. The results have been highly encouraging in many but not all cases, with durable responses in the limited time they have been followed up. The treatments carry some risks and have been reserved for refractory cases until now., Summary: High-dose therapy, without or with stem cell transplantation, is a valuable resource for the treatment of patients with refractory autoimmune neurologic diseases. It is important to define the diseases and patient characteristics likely to lead to benefit, to optimize the methods of treatment and to establish when in the patient's course to administer it. High-dose therapy may eventually become the standard for treatment of severe progressive autoimmune neurologic disorders.

Secondary myelodysplastic syndrome (MDS)/acute leukemia frequently evolves from severe aplastic anemia (SAA) following immunosuppressive therapy. Secondary clonal cytogenetic abnormalities have now been reported after noncytotoxic therapy in two additional settings: all trans retinoic acid (ATRA) treatment of acute promyelocytic leukemia (APL) and imatinib for chronic myeloid leukemia (CML). We propose that SAA, APL, CML, and MDS represent different manifestations of generalized insults to the bone marrow. In SAA, the insult to hematopoietic progenitors leads to an immune attack, while in APL, CML, and MDS, it gives rise to the malignant clones. A primary insult to bone marrow could simultaneously lead to several abnormal hematopoietic cell clones, with one dominating and the others present but below the level of detection. Such a 'field leukemogenic effect' would be analogous to the 'field cancerization effect' described in solid tumors. Nonspecific cytotoxic therapies, including antileukemic chemotherapy and allogeneic transplantation, have broad activity that could inhibit both the overt disease and other undetectable coexistent abnormal clones. In contrast, disease-specific targeted therapy such as immunosuppressive therapy in aplastic anemia, ATRA in APL, or imatinib in CML would have no activity against other abnormal clones, allowing them to expand and become detectable as the dominant clone declines.

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon, acquired disorder of blood cells caused by mutation of the phosphatidylinositol glycan class A (PIG-A) gene. The disease often manifests with haemoglobinuria, peripheral blood cytopenias, and venous thrombosis. The natural history of PNH has been documented in retrospective series; but there has only been one study that correlated the more sensitive and specific flow cytometric assays that have become available in the last decade with severe symptoms associated with PNH. In a retrospective analysis of 49 consecutive patients with PNH evaluated at Johns Hopkins, large PNH clones were associated with an increased risk for thrombosis as well as haemoglobinuria, abdominal pain, oesophageal spasm, and impotence. Of the 14 (29%) patients that developed thrombosis, nine died; six of these from complications related to thromboses. According to logistic regression modelling, for a 10% change in PNH clone size, the odds ratio for risk of thrombosis was estimated to be 1.64. No patient with <61% PNH granulocytes developed a thrombosis, whereas 12 of 22 patients (54.5%) with > or =61% PNH granulocytes manifested with thrombosis. These data not only confirm that the size of the PNH clone correlates with the risk for thrombosis, but they also suggest a correlation of PNH clone size to more symptomatic PNH.

Objective: The treatment options for patients with aplastic anemia who do not respond to conventional immunosuppression are limited. The aim of this study was to evaluate high-dose cyclophosphamide in patients with refractory severe aplastic anemia (SAA)., Materials and Methods: We treated 17 SAA patients with high-dose cyclophosphamide (50 mg/kg/day for 4 consecutive days) who previously did not respond to one or more courses of immunosuppressive therapy. Median age was 31 years (range 6-58); median disease duration was 14 months (range 6-58), and 8 patients met criteria for very severe aplastic anemia (absolute neutrophil count <0.2 x 10(9)/L) at the time of treatment., Results: At median follow-up of 29 months, 10 patients (59%) are alive. Nine patients (53%) achieved a drug-free remission after high-dose cyclophosphamide; 4 patients achieved a complete remission and 5 patients currently meet criteria for a partial remission but continue to improve. One nonresponder to high-dose cyclophosphamide developed paroxysmal nocturnal hemoglobinuria; another nonresponder developed a myelodysplastic syndrome. In responding patients, median time to 500 neutrophils was 54 days (range 35-119), median time to the last platelet transfusion was 99 days (range 51-751), and median time to the last red cell transfusion was 125 days (range 63-796)., Conclusion: High-dose cyclophosphamide shows promise for salvaging patients with refractory SAA.

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD -, and the isogenic cell line LD - PIGA + in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD - PIGA + cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab.

Multiple myeloma may be cured by myeloablative conditioning and allogeneic blood or marrow transplantation (alloBMT), but this occurs at the expense of high transplant-related mortality. In an endeavor to reduce procedure-related toxicity, this study retrospectively evaluated the safety, tolerability, and efficacy of T cell depletion by counterflow centrifugal elutriation before alloBMT. Fifty-one patients with stage II (6) or III (45) multiple myeloma received alloBMTs using T cell depletion by elutriation. Fifty-three percent (27 of 51) of patients had primary refractory disease at the time of transplantation, 10% (5 of 51) had relapsed disease, and 4% (2 of 51) had refractory relapsed disease. The median age was 49 (range, 32 to 62) years, and the median time from diagnosis to transplantation was 9 (range, 4 to 58) months. Patients had received a median of 1 (range, 1 to 3) regimen and 4 (range, 2 to 16) cycles of chemotherapy. In this population, transplant-related mortality rate was 24% (12 of 51) with 2 patients dying of graft-versus-host disease (GVHD). Thirty-one of 39 evaluable patients have experienced relapse, and the probability of progression-free survival 5 years after alloBMT alone is 16%. Sixteen patients were given donor lymphocyte infusions (DLI) at the time of relapse (n = 11) or for persistent disease 1 year after transplantation (n = 5). Acute or chronic GVHD was seen in 63% (10 of 16) of patients given DLI. Responses were seen in 8 of 16 patients (6 complete response [CR], 2 partial response [PR]) with 6 of 8 responding patients having GVHD. Five recipients of DLI remain in a continuous CR, ranging from 3 to 64 months in duration. Thus, like chronic myelogenous leukemia, allogeneic T cells appear to have potent antimyeloma activity that is critical for achieving a cure. DLI-induced remissions of multiple myeloma can be durable, even in patients with refractory multiple myeloma. Unlike chronic myelogenous leukemia, the antimyeloma effect of allogeneic T cells rarely occurs in the absence of clinically significant GVHD.

The progress of a female child with African type Fanconi anemia that evolves in time into paroxysmal nocturnal hemoglobinuria is described. Modern diagnostic methods are used to confirm this process. A discussion of possible mechanisms ensues.

Patients with myasthenia gravis (MG) who do not respond to conventional immunotherapeutic agents, or cannot tolerate their side effects, are considered "refractory." Ablation of the immune system followed by bone marrow transplant has been shown to cure experimental MG in rats. It is now known that immunoablative treatment with high-dose cyclophosphamide does not damage hematopoietic "stem cells," permitting repopulation of the immune system without bone marrow transplant. Recent evidence indicates that this treatment can induce durable remissions in autoimmune diseases. We treated three myasthenic patients, for whom treatment with thymectomy, plasmapheresis, and conventional immunotherapeutic agents failed, by using high-dose cyclophosphamide (50mg/kg/day intravenously for 4 days) followed by granulocyte colony stimulating factor. All three patients tolerated the treatment well and have had marked improvement in myasthenic weakness, permitting reduction of immunosuppressive medication to minimal levels. Acetylcholine receptor (AChR) antibody levels decreased in two AChR antibody-positive patients, and anti-MuSK antibody levels decreased in one "AChR antibody-negative" patient. The patients have been followed for up to 3.5 years, with no recurrence of symptoms. High-dose cyclophosphamide treatment appears to be an effective and safe treatment for selected patients with refractory MG. Further follow-up of these and additional patients will be needed to determine whether the benefit is durable.

Objective: High-dose chemotherapy followed by hematopoietic stem cell transplantation is increasingly being studied as a treatment for severe autoimmune disorders, such as systemic lupus erythematosus (SLE). High-dose cyclophosphamide, the foundation of virtually all conditioning regimens for stem cell transplantation, is not myeloablative; therefore, when high-dose cyclophosphamide is used alone, autografting, with its potential for reinfusing autoreactive effector cells, is not required. We undertook this study to investigate the safety and efficacy of high-dose cyclophosphamide without stem cell transplantation in refractory SLE., Methods: We treated 14 patients with moderate-to-severe SLE that had been refractory to corticosteroids and one or more additional immunosuppressive drugs. All patients received 50 mg/kg of cyclophosphamide for 4 consecutive days followed by 5 microg/kg granulocyte colony-stimulating factor until the neutrophil count was 1 x 10(9)/liter for 2 consecutive days. Patients were followed up monthly for disease activity using the physician's global assessment, SLE Disease Activity Index, and assessment of functioning of involved organs. The Responder Index for Lupus Erythematosus was used to define partial or complete response., Results: The median time to achieve a neutrophil count of 0.5 x 10(9)/liter was 14 days (range 11-22 days) after the last dose of cyclophosphamide. Patients required a median of 2 units (range 2-5) of packed red blood cells, and a median of 16 days (range 0-23 days) elapsed from the last dose of cyclophosphamide to the last platelet transfusion. There were no deaths or fungal infections. Significant improvements in physician's global assessment (mean difference 1.4; P < 0.0001), SLE Disease Activity Index (mean difference 4.1; P = 0.0039), and prednisone dosage (mean difference 14.9 mg/day; P = 0.002) were observed. Responses, including 5 durable complete responses, were observed in all organ systems (renal, central nervous system, and skin) with involvement that had led to patient enrollment., Conclusion: High-dose cyclophosphamide without stem cell transplantation leads to rapid hematopoietic reconstitution and has significant clinical benefit in patients with refractory SLE. Therefore, this approach deserves further study.

High-dose cyclophosphamide, without stem cell rescue, has been used successfully to treat aplastic anemia and other autoimmune disorders. To determine the safety and efficacy of high-dose cyclophosphamide among patients with severe refractory autoimmune hemolytic anemia, we treated 9 patients with cyclophosphamide (50 mg. kg(-1). d(-1) for 4 days) who had failed a median of 3 (range, 1-7) other treatments. The median hemoglobin before treatment was 6.7 g/dL (range, 5-10 g/dL). The median time to reach an absolute neutrophil count of 500/microL or greater was 16 days (range, 12-18 days). Six patients achieved complete remission (normal untransfused hemoglobin for age and sex), and none have relapsed after a median follow-up of 15 months (range, 4-29 months). Three patients achieved and continue in partial remission (hemoglobin at least 10 g/dL without transfusion support). High-dose cyclophosphamide was well tolerated and induced durable remissions in patients with severe refractory autoimmune hemolytic anemia.

High-dose cyclophosphamide (200 mg/kg) was initially developed as a conditioning regimen for allogeneic bone marrow transplantation. Recently, high-dose cyclophosphamide without bone marrow transplantation has been employed as a method to induce durable treatment-free remissions in severe aplastic anemia and a variety of other severe autoimmune disorders. The premise underlying this approach is that high-dose cyclophosphamide is maximally immunosuppressive, but not myeloablative. Early hematopoietic stem cells are spared the cytotoxicity of cyclophosphamide because of their high levels of aldehyde dehydrogenase, an enzyme that confers resistance to the drug. Conversely, autoimmune effector cells (T cells, B cells, and NK cells) are exquisitely sensitive to high-dose cyclophosphamide because of their relatively low levels of aldehyde dehydrogenase. Intensive investigation is underway to determine which autoimmune disorders will most benefit and where in the natural history of these diseases to employ this rapidly developing therapy.

Between January 1987 and January 1997, 69 eligible patients with acute myeloid leukaemia (AML) in either second (CR2) or third (CR3) complete remission (CR2 = 60, CR3 = 9) underwent 4-hydroperoxycyclophosphamide-purged autologous bone marrow transplantation (BMT) at the Johns Hopkins Oncology Center. The patients' median age was 27 years (range 1-62) and all received busulphan and cyclophosphamide as their preparative regimen. The probability of event-free survival (EFS) at 5 years was 30% [95% Confidence Interval (CI): 19-42%] for CR2 patients and 22% (3-51%) for those in CR3, with a median follow up of 8 years in the surviving group. The median time to an absolute neutrophil count of 0.5 x 109/l was 45 d (range 20-185). Relapse was the major cause of failure with a relapse rate of 55% in CR2 and 44% in CR3, while the non-relapse, transplant-related mortality rate was 15% in CR2 and 33% in CR3. In univariate analysis, patient age, cytogenetics, white blood cell count at presentation, CR1 duration and the sensitivity of clonogeneic leukaemia (CFU-L) in the graft to 4HC were all prognostic for EFS. Using each of these significant variables in multivariate modelling, patient age and sensitivity of CFU-L to 4HC were determined to be predictors of EFS. 4HC-purged autologous BMT produced results similar to allogeneic BMT for AML patients beyond first remission.

Bryostatin-1 inhibits acute myeloid leukemia (AML) in vitroat doses that stimulate the growth of normal hematopoietic progenitors.Although bryostatin-1 has a number of distinct biological activities, those specifically responsible for its antileukemic activity are unclear. We found that bryostatin-1 (10(-8) M) inhibited cell cycling at G(1), induced phenotypic evidence of differentiation, and limited the clonogenic growth of both AML cell lines and patient specimens. This activity was markedly enhanced by granulocyte/macrophage-colony stimulating factor, whereas growth factor-neutralizing antibodies completely inhibited both the differentiating and antileukemic activities of bryostatin-1. Cell cycle inhibition and growth factors were also required for the differentiating activities of two unrelated agents, hydroxyurea and phenylbutyrate. These data suggest that many pharmacological differentiating agents require both cell cycle arrest and lineage-specific growth factors for full activity and may explain why these agents have demonstrated only limited clinical efficacy.

High-dose cyclophosphamide was developed as a conditioning regimen for allogeneic bone marrow transplantation. Later, it was discovered that high-dose cyclophosphamide spares early hematopoietic stem cells because of their relatively high levels of the enzyme aldehyde dehydrogenase; thus, high-dose cyclophosphamide is a potent immunosuppressive agent, but nonmyeloablative. Recent reports demonstrate that high-dose cyclophosphamide without bone marrow transplantation induces durable treatment-free remissions in severe aplastic anemia and a variety of other autoimmune disorders; however, there is lingering concern about the safety of this approach.

Purpose: To evaluate the long-term outcome after allogeneic (allo) and autologous (auto) blood or marrow transplantation (BMT) in patients with relapsed or refractory Hodgkin's lymphoma (HL)., Patients and Methods: We analyzed the outcome of 157 consecutive patients with relapsed or refractory HL, who underwent BMT between March 1985 and April 1998. Patients

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming protein aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before exposure to aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in aerolysin. The inactive fluorescent proaerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

Background: Severe aplastic anemia is a life-threatening bone marrow failure disorder. High-dose cyclophosphamide therapy followed by allogeneic bone marrow transplantation cures the disease. However, it requires a suitable donor and carries the risk for graft-versus-host disease. A small pilot study demonstrated that high-dose cyclophosphamide therapy without bone marrow transplantation leads to durable, treatment-free complete remission., Objective: To confirm the safety and efficacy of high-dose cyclophosphamide therapy alone in patients with severe aplastic anemia., Design: Uncontrolled clinical trial., Setting: Three tertiary care hospitals., Patients: 19 patients with untreated severe aplastic anemia., Intervention: Cyclophosphamide, 50 mg/kg of body weight per day for 4 consecutive days., Measurements: Probability of response and overall survival were measured. Complete remission was defined as normal blood count for age and sex. Partial remission was defined as independence from transfusion and an absolute neutrophil count greater than 0.5 x 10(9) cells/L without growth factor support. Nonresponders were patients who remained transfusion dependent or died. Relapse was defined as no longer meeting criteria for partial or complete remission., Results: The median time to an absolute neutrophil count of 0.5 x 10(9) cells/L was 49 days. The probability of survival was 84% (95% CI, 59% to 95%) at 24 months. The probability of achieving treatment-free remission was 73% (CI, 51% to 91%) at 24 months, and the probability of achieving complete remission was 65% (CI, 39% to 89%) at 50 months. No responding patients have had relapse or have developed secondary clonal disorders., Conclusions: High-dose cyclophosphamide therapy without bone marrow transplantation produces durable treatment-free remission in severe aplastic anemia. This approach deserves further study in patients with severe aplastic anemia who are not suitable candidates for allogeneic bone marrow transplantation.

Background: Alloimmunization is a major problem for patients being considered for solid organ transplantation and in patients who require blood transfusion support. We previously demonstrated that high-dose cyclophosphamide (200 mg/kg) without hematopoietic stem cell transplantation leads to durable complete remissions in aplastic anemia and other autoimmune disorders. We now examine the ability of high-dose cyclophosphamide to eliminate alloreactivity., Methods: IgG-specific antibodies to HLA class I were assayed using enzyme-linked immunosorbent assays in 18 consecutive patients with severe aplastic anemia before and after treatment with high-dose cyclophosphamide., Results: Anti-HLA antibodies were detected before or shortly after therapy in 5 of the 18 patients studied. Complete remission of aplastic anemia was achieved in four of these five patients. High-dose cyclophosphamide markedly reduced anti-HLA antibody titers in these four patients; they were completely eradicated in three patients. Only one patient did not achieve significant reduction in the alloantibody titer after high-dose cyclophosphamide., Conclusions: High-dose cyclophosphamide without stem cell transplantation can eradicate HLA-specific alloantibody.

Phosphatidylinositol glycan-A (PIGA) is a gene that encodes an element required for the first step in glycosylphosphatidylinositol (GPI) anchor assembly. Because PIGA is X-located, a single mutation is sufficient to abolish cell surface GPI-anchored protein expression. In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator (Mut) phenotypes in cancer. We examined eight Mut colon cancer lines and four non-Mut colon cancers as controls. In every case, flow cytometric analyses of cells sorted for low fluorescence after staining for GPI-linked CD59 and CD55 revealed negative peaks in the Mut lines but not in the controls. Single cell cloning of purged and sorted GPI-anchor- HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations. Pretreatment of the Mut lines with anti-CD55 or anti-CD59 antibodies and complement or with the GPI-anchor-reactive bacterial toxin aerolysin enriched for the GPI-anchor- populations. Expansion of purged GPI-anchor+ cells in the Mut lines and analyses using aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10(-5) to 10(-4), values similar to the mutation frequencies of the hprt gene. This novel approach allows for the detection of as yet undescribed repair or replication defects and in addition to its considerably greater ease of use than existing techniques and in principle would not require the production of cell lines.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Many institutions worldwide are conducting clinical studies using immunoablative therapy followed by hematopoietic stem cell transplantation for the treatment of SLE. Interpretation of these studies will be complicated by the differences in patient selection, conditioning regimens, and the method of stem cell collection. A major concern with this approach is that autoreactive effector cells will be re-infused with the autologous graft. The recent demonstration that immunoablative therapy (cyclophosphamide 200 mg/kg) can be safely delivered without the need for stem cell rescue offers a potential way to circumvent this problem. Early results employing immunoablative therapy, with or without stem cell rescue, are encouraging; however, longer follow-up and additional patients are necessary to validate this approach.

Aerolysin is a channel-forming toxin that binds to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on target cells. Here, we show that subnanomolar concentrations of aerolysin trigger apoptosis of T lymphomas. Using inactive aerolysin variants, we determined that apoptosis was not directly triggered by binding to GPI-anchored receptors, nor was it caused by receptor clustering induced by toxin oligomerization. Apoptosis was caused by the production of a small number of channels in the cell membrane. Channel formation resulted in a rapid increase in intracellular calcium, which may have been the signal for apoptosis. Overexpression of the antiapoptotic protein bcl-2 blocked aerolysin-induced apoptosis, although this effect was overcome at higher toxin concentrations.

Paraneoplastic pemphigus (PNP) is a refractory and life-threatening autoimmune mucocutaneous disease. We have recently reported the effectiveness and safety of ablative intravenous cyclophosphamide (200 mg/kg daily over 4 days) without stem cell rescue in patients with refractory autoimmune diseases including systemic lupus erythematosus, autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, and aplastic anemia. We report chronic lymphocytic leukemia-associated PNP in a patient who presented with extensive and debilitating painful oral ulcerations and received ablative therapy. The patient tolerated the regimen well and showed a slow but sustained improvement despite persistence of the underlying neoplasm. Eighteen months after therapy, the oral ulcerations were almost completely healed and the circulating autoantibodies became negative. Currently, the patient remains on cyclosporine and a low dose of prednisone. This provides further evidence for the efficacy and safety of this regimen in the management of severe autoimmune diseases including PNP.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Autoimmune diseases afflict approximately 2% of the US population and span virtually every medical specialty. Most patients with autoimmune diseases have a normal life expectancy and are managed conservatively; however, a subset of patients have a progressive disease course associated with significant morbidity and mortality. High-dose cytotoxic therapy followed by autologous stem cell transplantation has been proposed as a novel treatment for severe autoimmune diseases. This review will focus on recent advances in the immunopathogenesis of autoimmunity and will review preliminary results of clinical trials employing hematopoietic stem cell transplantation for the treatment of autoimmunity.

Autologous bone marrow transplantation (BMT) has not been curative in chronic myeloid leukemia (CML), because of the inability to purge CML from the autograft and the absence of the allogeneic T cell-mediated antileukemic activity. However, recent advances demonstrate that normal progenitors can be selected from CML marrows by a variety of techniques, including isolation by their small size. Furthermore, we found that myeloid growth factors have a potent antileukemic effect against CML progenitors in vitro by inducing their terminal differentiation. Based on these data, we initiated a trial of autologous BMT in patients with high-risk CML. Autografts were processed in an attempt to enrich for normal progenitors, first by isolating small cells by counterflow centrifugal elutriation and then incubating them in granulocyte-macrophage colony-stimulating factor (GM-CSF) for 72 hours. After a conditioning regimen of busulfan and cyclophosphamide, all patients received GM-CSF daily for 2 months. The median age of the 13 patients in the trial was 45 years (range 17-56 years). The median duration of disease before BMT was 24 months (range 13-72 months). Eight patients were in chronic phase (CP), and five were in accelerated phase (AP). All patients failed to achieve a cytogenetic response to interferon-alpha and were 100% Philadelphia chromosome (Ph)+ before BMT. There were three transplant-related deaths, all AP patients. All of the remaining 10 patients engrafted with some degree of Ph- hematopoiesis; despite high-risk features, nine patients engrafted 100% Ph-. All patients relapsed cytogenetically at a median of 6 months (range 4-22 months). These results demonstrate that autologous BMT can consistently induce complete Ph- engraftment in CP patients. GM-CSF appears to produce a clinical antileukemic effect against CML after autologous BMT.

Background: Immunoablative high-dose cyclophosphamide without stem-cell rescue induces durable, complete remission in most patients with aplastic anemia., Objective: To determine the efficacy of high-dose cyclophosphamide in various refractory, severe autoimmune diseases., Design: Prospective phase II study., Setting: Johns Hopkins University (Baltimore, Maryland) and Hahnemann University (Philadelphia, Pennsylvania)., Patients: Eight patients with refractory, severe autoimmune disease., Intervention: Immunoablative high-dose cyclophosphamide (50 mg/kg of body weight per day) for 4 consecutive days., Measurements: Clinical and laboratory variables of autoimmune disease., Results: Seven patients improved markedly: Five achieved complete remission and two achieved partial remission. Four patients have remained in continuous complete remission for 3 to 21 months, and two patients in partial remission continue to improve after 14 and 19 months of follow-up. High-dose cyclophosphamide was well tolerated; median times to a neutrophil count of 0.5 x 10(9) cells/L and platelet transfusion independence were 17 and 16 days, respectively., Conclusions: Immunoablative high-dose cyclophosphamide without stem-cell rescue can induce complete remission in patients with refractory, severe autoimmune disease. Reemergence of marrow function is similar to that seen after autologous transplantation and does not carry the risk for reinfusion of autoaggressive lymphocytes with the autograft.

Despite extensive investigation into mechanisms of drug resistance in acute myeloid leukaemia (AML), the aetiology of therapeutic resistance is unclear. We found that five leukaemia cell lines (K562, HL-60, CEM. CEM induced to overexpress bcl-2, and REH) displayed parallel sensitivity to four antileukaemia drugs with different mechanisms of action, with K562 generally being the least sensitive and REH being the most sensitive. The amount of spontaneous apoptosis in the cell lines after serum-free culture paralleled their drug sensitivity: K562 cells displayed the least apoptosis at 24h (2.50 +/- 0.24%) and REH the most (24.47 +/- 8.22%). The extent of spontaneous apoptosis of leukaemic blasts from 39 patients with newly diagnosed de novo AML also correlated with the success of the intensive, infusional cytarabine-based induction therapy. There was a median of 19.5% (range 3.6-64%) apoptotic AML cells after 24 h of serum-free culture in patients who entered a complete remission compared with 4.2% (1.8-7.0%) apoptotic AML cells in patients who did not achieve a complete remission (P = 0.0007). Thus, inhibited apoptosis was associated with both in vitro and in vivo pan-resistance to antileukaemic chemotherapy. The cause of inhibited apoptosis in AML is probably a function of interactions among multiple signals that influence apoptosis. Assessment of spontaneous apoptosis may serve as an important prognostic factor for AML.

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder characterized by pancytopenia and a hypocellular marrow. Drugs, chemical exposure, radiation, and viruses are implicated as etiologic agents, although the majority of community-acquired SAA is idiopathic. Regardless of the inciting event, most cases of SAA result from immune-mediated destruction of bone marrow progenitor cells, which spares pluripotent hematopoietic stem cells. SAA is treated by either allogeneic bone marrow transplantation (BMT) or immunosuppressive therapy. BMT restores normal hematopoiesis and cures the disease in 60% to 80% cases, with the major causes of failure being graft rejection and graft-versus-host disease. Most patients treated with immunosuppressive therapy recover hematopoiesis sufficiently to not require transfusions and are free of infection, although in many, recovery is incomplete. Moreover, up to 50% of SAA patients successfully treated with immunosuppressive therapy relapse or develop a secondary clonal disorder, such as paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, or leukemia. High-dose cyclophosphamide without BMT is capable of restoring normal hematopoiesis with little or no risk of relapse or secondary clonal disorders. A number of effective treatment options for the treatment of SAA are now available. The optimal approach for definitive management of SAA continues to evolve.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene, PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found that PIG-A mutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34(+) (CD59(-)) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIG-A gene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting from PIG-A mutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.

The National Cancer Institute (NCI) recently alerted clinicians to the possibility that patients, entered on a NCI-sponsored cooperative group trial of doxorubicin and cyclophosphamide adjuvant therapy for breast cancer, may be at high risk of developing secondary acute myeloid leukemia (AML). Secondary AML following standard doses of doxorubicin and cyclophosphamide is uncommon, suggesting that the high risk on this trial may result from its higher-than-standard doses of chemotherapy. However, the cases of secondary AML were characteristic of the type that follows treatment with topoisomerase II-active agents, especially etoposide, and this type of secondary AML is rare after treatment with either cyclophosphamide or doxorubicin at any dose. We raise the possibility that another component of this trial, hematopoietic growth factors to decrease the toxicities related to myelosuppression, may play an important role in the development of secondary AML. Growth factors not only stimulate hematopoietic progenitor proliferation and differentiation, they also regulate hematopoietic cell survival by interfering with apoptosis (programmed cell death). Inhibition of apoptosis by a variety of genetic factors is an important mechanism of oncogenesis, and appears to be the initiating event in some malignancies. Growth factor-mediated suppression of the apoptotic death of hematopoietic progenitors damaged by chemotherapy may contribute to their leukemic transformation.

The paper presents a review of the results obtained by the authors on the study of external (gamma) and internal (I-131) radiation effects on the functional morphology and linkage of the diffuse neuroendocrine system (DNES) and amine precursor uptake and decarboxylation (APUD) cells of the stomach and duodenum. The investigations performed enabled us to determine that the morphological changes noted in APUD cells had a dose and time dependency. The present study supports the point of view that the radiation initiates serotonin release from APUD cells, which appears to initiate the mechanism of early postirradiation dysfunctions of the gastrointestinal tract and the subsequent adaptive response of DNES. Analysis of our results, together with a review of the literature, indicates that APUD cells actively participate both in pathogenesis of radiation injury and development of organ and tissue radiosensitivity.

CD4 is the major cellular receptor for the human immunodeficiency virus (HIV). A hybrid gene encoding the extracellular domains of CD4, linked to the sequence encoding the membrane attachment region of the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF) was stably transfected into HeLa cells. The resultant cell line (T4HD) expressed GPI-anchored CD4DAF at high levels and was susceptible to gene transfer with a recombinant HIV vector. In an effort to expand the spectrum of cells susceptible to HIV gene transfer, CD4DAF was released from the surface of the T4HD cell line by detergent lysis, purified by immunoaffinity chromatography, and reincorporated into native HeLa cells. Incorporation occurred via the GPI anchor as evidenced by cleavage with phosphatidylinositol-specific phospholipase C. More than 95% of the CD4DAF-treated HeLa cells were CD4-positive by flow cytometry, and kinetic analysis demonstrated that over 75% of the fusion protein remained anchored to the cell membrane after 90 min at 37 degrees C. The purified protein retained its ability to bind the envelope protein of HIV. When incorporated, it bound fluorescein isothiocyanate (FITC)-conjugated gp120, and in its soluble form blocked transduction of CD4-positive cells incubated with an HIV-derived vector containing the Neo gene. In contrast to the T4HD cells, exposure of CD4DAF-treated cells to the Neo HIV vector yielded only transient neomycin-resistant colonies. These results suggest that endogenous synthesis of the CD4 molecule may be necessary for successful HIV genomic integration.

We report a case of acquired sideroblastic anaemia precipitated by progesterone. On two separate occasions, over 15 years apart, the patient developed sideroblastic anaemia with iron overload shortly after the administration of progesterone. No other cause for sideroblastic anaemia was found, and treatment with folic acid, pyridoxine or androgens corrected the anaemia. In both instances removal of the progestational agent led to prompt disappearance of the anaemia as well as the ringed sideroblasts. Using a two-phase liquid culture procedure in which human peripheral blood-derived progenitor cells undergo erythroid proliferation and differentiation, we demonstrated enhanced sensitivity of the patient's erythroid progenitors to progesterone. We conclude that progesterone should be added to the list of medications known to be associated with acquired sideroblastic anaemia.