Long-term systemic lupus erythematosus disease control after allogeneic bone marrow transplantation.

Systemic lupus erythematosus (SLE), a disorder of the immune system, is potentially curable by allogeneic bone marrow transplantation (alloBMT). Until recently, alloBMT was limited by donor availability and toxicity. Reduced intensity conditioning (RIC) combined with post-transplantation cyclophosphamide (PTCy) has improved the availability and safety of alloBMT permitting its exploration in severe-refractory autoimmune illnesses. We report the six-year follow-up of a young female whose refractory SLE-associated nephrosis resolved after RIC alloBMT with PTCy.

Changing prognosis in paroxysmal nocturnal haemoglobinuria disease subcategories: an analysis of the International PNH Registry.

BACKGROUND: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare disease. Although much progress has been made in the understanding of the pathophysiology of the disease, far less is known with respect to the clinical outcomes of patients with PNH. Few retrospective studies provide survival estimates, and even fewer have explored the clinical heterogeneity of the disease. Haemolytic and aplastic anaemia (AA) forms of the disease have been recognised as main disease categories, with the haemolytic form being associated with the worst prognosis by the largest studied cohort some years ago. AIMS: To describe mortality and causes of death in PNH overall and by PNH classification and to evaluate risk factors associated with mortality. METHODS: We analysed data of 2356 patients enrolled in the International PNH Registry with multivariate analyses, using time-dependent covariates. Patients were classified into haemolytic, AA/PNH syndrome or intermediate PNH. RESULTS: Overall, 122 (5.2%) patients died after enrolment, the incidence according to subcategories being 5.1, 11.7, 2.0 and 4.8% for patients with haemolytic PNH, AA-PNH, intermediate and insufficient data respectively. Older age and decreased performance status also affected survival in multivariate analysis. Improved outcome of patients with haemolytic PNH suggests that eculizumab treatment in PNH may be associated with improved survival. CONCLUSION: A detailed analysis of clinical presentations and causes of death in patients with PNH, overall and by disease subcategories, provide evidence that in the current era, patients with haemolytic PNH are no longer those who harbour the worst prognosis. This finding differs sharply from what has been previously reported.

Immunologic recovery following autologous stem-cell transplantation with pre- and posttransplantation rituximab for low-grade or mantle cell lymphoma.

BACKGROUND: Rituximab may improve transplant outcomes but may delay immunologic recovery. PATIENTS AND METHODS: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m(2) was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed. RESULTS: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1-2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%. CONCLUSION: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.

High-dose cyclophosphamide without stem cell rescue in scleroderma.

OBJECTIVE: To investigate the safety and tolerability of high-dose cyclophosphamide without stem cell rescue in scleroderma. METHODS: An open-label, single-site, uncontrolled study design entered patients with active diffuse cutaneous scleroderma. Patients were treated with cyclophosphamide (50 mg/kg) intravenously daily for 4 consecutive days (total 200 mg/kg) followed by granulocyte colony-stimulating factor (5 microg/kg/day). The primary clinical efficacy end point was the modified Rodnan skin score (mRSS). Secondary end points included the Health Assessment Questionnaire-Disability Index (HAQ-DI), physician global assessment (PGA) and pulmonary function tests. RESULTS: Six patients (4 men, 2 women) aged 19-60 years were entered into the study. One patients died early in the protocol, thus five patients had follow-up data. The percentage reduction of the mRSS in these five evaluable patients within 1 month of treatment was 60%, 55%, 41%, 31% and 0%. The patient with no decline in skin score at 1 month showed a decrease in skin score from 41 to 26 by the 3-month visit, a 37% improvement. Three patients sustained the improvement after treatment for 24, 12 and 12 months. Two patients relapsed at 12 and 6 months after treatment. The PGA and HAQ-DI scores improved in five of the six patients by 72% and 79% respectively at 3 months. The only serious adverse event was a death that occurred owing to infection after neutrophil count recovery. CONCLUSIONS: High-dose cyclophosphamide without stem cell rescue can lead to a clinically significant improvement in skin score and measures of disease severity in patients with diffuse cutaneous scleroderma.

Quantitative analysis of bone marrow CD34 cells in aplastic anemia and hypoplastic myelodysplastic syndromes.

Aplastic anemia (AA) and hypoplastic myelodysplastic syndromes (hMDS) are often difficult to distinguish. However, an accurate diagnosis is important because the prognosis and treatment of these diseases may differ. CD34+ hematopoietic progenitors are central to the pathogenesis of both disorders; they are the targets of the autoimmune attack in AA and neoplastic transformation in MDS. The aim of this study was to assess whether bone marrow CD34+ cell numbers could be used in differentiating between AA and hMDS. The percentage of bone marrow CD34+ cells was normal or increased (mean -3.5+0.5%, range 1-7%) in 15 of 35 patients studied, and low (mean -0.13 +/- 0.02%, range 0.02-0.36%) in 20 of 35 patients. All patients with a normal or increased percentage of CD34+ cells were ultimately diagnosed with hMDS based on the detection of clonal cytogenetic abnormalities or progression to refractory anemia with excess blasts/acute myeloid leukemia. All patients with low marrow CD34+ cell numbers met standard clinical criteria for AA and have not demonstrated neoplastic transformation with follow-up. Quantification of marrow CD34+ cells may serve as an important tool for distinguishing between AA and hMDS.

Transplant-associated thrombotic microangiopathy: opening Pandora's box.

Transplant-associated thrombotic microangiopathy (TA-TMA) is an early complication of hematopoietic cell transplantation (HCT). A high mortality rate is documented in patients who are refractory to calcineurin inhibitor cessation. Estimates of TA-TMA prevalence vary significantly and are higher in allogeneic compared with autologous HCT. Furthermore, our understanding of the pathophysiology that is strongly related to diagnosis and treatment options is limited. Recent evidence has linked TA-TMA with atypical hemolytic uremic syndrome, a disease of excessive activation of the alternative pathway of complement, opening the Pandora's box in treatment options. As conventional treatment management is highly inefficient, detection of complement activation may allow for early recognition of patients who will benefit from complement inhibition. Preliminary clinical results showing successful eculizumab administration in children and adults with TA-TMA need to be carefully evaluated. Therefore, realizing the unmet needs of better understanding TA-TMA in this complex setting, we aimed to summarize current knowledge focusing on (1) critical evaluation of diagnostic criteria, (2) epidemiology and prognosis, (3) recent evidence of complement activation and endothelial damage and (4) treatment options.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Glycophosphatidylinositol-anchored protein deficiency as a marker of mutator phenotypes in cancer.

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.

Long-term results of blood and marrow transplantation for Hodgkin's lymphoma.

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

Riddle: what do aplastic anemia, acute promyelocytic leukemia, and chronic myeloid leukemia have in common?

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.

Are growth factors leukemogenic?

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.

Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer.

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.

Elimination of alloantibodies by immunoablative high-dose cyclophosphamide.

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.

Bone marrow transplantation in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome is a rare autosomal recessive disorder characterized by exocrine pancreatic dysfunction, metaphyseal dysostosis and bone marrow dysfunction with a predilection towards severe hematologic complications. Allogeneic bone marrow transplantation has been used as a therapeutic approach for SDS patients with serious hematologic abnormalities with mixed results. There is some concern that these patients may be more susceptible to early (<100 days) transplant-related complications than other transplant groups. We report a patient who received a matched allogeneic transplant without developing serious early transplant-related complications, but eventually died from relapse of his disease. Although experience is limited, a review of the reported cases suggests patients with SDS may be transplanted without significant short-term morbidity and mortality.

Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.

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.

Hematopoietic stem cell transplantation for systemic lupus erythematosus.

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.

Frequency of veno-occlusive disease of the liver in bone marrow transplantation with a modified busulfan/cyclophosphamide preparative regimen.

Veno-occlusive disease (VOD) of the liver is a major complication of bone marrow transplantation (BMT). The overall frequency of VOD has ranged from 20 to 30%, with a mortality rate greater than 40%, as reported by centers utilizing cyclophosphamide (Cy) and total body irradiation (CyTBI) or various chemotherapeutic regimens, including the busulfan (Bu) (4 mg/kg for 4 days) and Cy (50 mg/kg for 4 days) (BuCy4) combination. Since 1986, Hahnemann University (HU) has primarily used the BuCy2 regimen, i.e., Bu (4 mg/kg for 4 days) followed by Cy (60 mg/kg for 2 days). We reviewed 74 consecutive patients who received either an autologous or allogeneic BMT for various malignancies from January 1986 through October 1988 to determine the frequency of VOD. Seven of 74 consecutive patients met clinical criteria for VOD, for a total frequency of 9.5%. Fifty-five patients were conditioned with various other regimens. Only 5 of the patients conditioned with BuCy2 developed VOD (9.1%). This is less than the 25% reported frequency of VOD in patients who received CyTBI (1,000 rads) and less than the 24% reported frequency of VOD in patients who received BuCy4. Only one of seven patients who developed VOD died from the disease. One patient died of sepsis after the VOD had almost completely resolved. The remaining five completely recovered. We conclude that the total Cy dose, and not Bu, is the major factor in the occurrence of VOD in Bu/Cy BMT preparative regimens, and the BuCy2 regimen reduces the frequency of VOD in autologous and allogeneic graft recipients when compared to CyTBI or the BuCy4 regimens.

Low-dose caudal morphine for postoperative analgesia in infants and children: a report of 500 cases.

STUDY OBJECTIVE: To determine the effectiveness of morphine 0.03 mg/kg or 0.04 mg/kg administered caudally to children for postoperative pain relief. DESIGN: Retrospective chart review. SETTING: University-affiliated hospital. PATIENTS: The charts of 500 children who had undergone various surgical procedures and who were given caudal morphine 0.03 mg/kg or 0.04 mg/kg either prior to the surgical procedure or immediately at the conclusion of the surgical procedure. MEASUREMENTS AND MAIN RESULTS: Parameters of respiratory rate, oxygen saturation, nausea and vomiting, voiding problems, and pruritus were recorded for each patient. There was no respiratory depression noted in the review of the 500 patients; 23% had nausea and vomiting, 3% had voiding problems needing bladder catheterization, and 7% reported pruritus, which was treated with either diphenhydramine or naloxone. CONCLUSION: Statistically there were no differences between morphine 0.03 mg/kg and morphine 0.04 mg/kg in all the study parameters. There was no respiratory depression in the 500 cases reviewed. The postoperative pain relief ranged from 6 hours to over 24 hours. This method of immediate postoperative pain management proved to be effective and safe.

The diagnostic content of family practice: 50 most common diagnoses recorded in the WAMI community practices.

Because there are several methodological deficiencies in previously published studies, a prospective study was carried out of the 50 most common diagnoses of community-based family practices in the Pacific Northwest. Age and sex of patients and reliability of data were controlled. The data reported show reasonable concordance with other published accounts for 10 most common diagnostic categories. For less frequent diagnoses, however, high variability in rank order is the rule, both within this study and by comparison with other studies. This suggests that the diagnostic content of family practice is far from universal and that diagnostic idiosyncrasies of physicians, regional differences in rates of disease, practice style, and as yet other unexplained factors may significantly influence the diagnostic content of family practice.

A role for the extended amygdala in the fear-enhancing effects of acute selective serotonin reuptake inhibitor treatment.

Selective serotonin reuptake inhibitors (SSRIs) are reported to exacerbate symptoms of anxiety when treatment is initiated. These clinical findings have been extended to animal models wherein SSRIs also potentiate anxiety and fear learning, which depend on the amygdala. Yet, little is known about the role of specific amygdalar circuits in these acute effects of SSRIs. Here, we first confirmed that a single injection of fluoxetine 1 h before auditory fear conditioning potentiated fear memory in rats. To probe the neural substrates underlying this enhancement, we analyzed the expression patterns of the immediate early gene, Arc (activity-regulated cytoskeleton-associated protein). Consistent with previous reports, fear conditioning induced Arc protein expression in the lateral and basal amygdala. However, this was not enhanced further by pre-treatment with fluoxetine. Instead, fluoxetine significantly enhanced expression of Arc in the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Next, we tested whether direct targeted infusions of fluoxetine into the CeA, or BNST, leads to the same fear-potentiating effect. Strikingly, direct infusion of fluoxetine into the BNST, but not the CeA, was sufficient to enhance fear memory. Moreover, this behavioral effect was also accompanied by robust Arc expression in the CeA, similar to the systemic injection. Our results identify a novel role for the BNST in the acute fear-enhancing effects of SSRIs. These findings highlight the need to look beyond the traditional focus on input nuclei of the amygdala and add to accumulating evidence implicating these microcircuits in gating fear and anxiety.