Pharmacokinetics and clinical activity of very low-dose alemtuzumab in transplantation for acute leukemia.

The optimal dose of in vivo-administrated alemtuzumab in the allogeneic transplantation setting has not been defined. We report our experience on 37 patients with high-risk diseases, mainly acute leukemia (AML 23, ALL 10 patients), who underwent sibling (49%) or unrelated (51%) PBSCT (35 patients), and received a total dose of only 10-20 mg Campath-1H as part of the conditioning, and post-transplant CYA without MTX. The neutrophil and especially the platelet engraftment were rapid. There were only two grade III-IV acute GvHD cases, which occurred in unrelated transplants in the Campath-10 cohort. Chronic GvHD developed in six cases (17%) and was limited to skin in five of them. After a median follow-up of 371 days (59-1191), 70% patients are alive and in CR (Karnofsky 100%), and 11 died (TRM n=6, relapse n=5). From the five patients relapsed, three were at advanced stage at transplant and four underwent sibling HCT with the higher (20 mg) alemtuzumab dose. With the 10 mg alemtuzumab schedule (5 mg/day at days -2 and -1) we achieve at day of transplantation low but still lymphotoxic alemtuzumab serum concentrations (176 ng/mL), whereas levels declined fast thereafter, and at engraftment nearly no Campath antibody remained in the patient's serum.

Human herpesvirus 6-related pure red cell aplasia, secondary graft failure, and clinical severe immune suppression after allogeneic hematopoietic cell transplantation successfully treated with foscarnet.

Human herpesvirus 6 (HHV-6) is frequently detected after allogeneic hematopoietic cell transplantation (allo-HCT); however, the clinical interpretation of HHV-6 viremia in a transplant patient is challenging as it may signify asymptomatic reactivation, chromosomal integration of the virus genome in the donor or recipient with no clinical significance, or severe HHV-6 disease. Here we present a case of HHV-6 disease after allo-HCT presenting as pure red cell aplasia, secondary graft failure, and severe immunosuppression causing multiple severe bacterial super-infections. Examination of pre-transplant patient and donor samples as well as serial determination of HHV-6 DNA copy numbers after transplantation were necessary to definitively interpret HHV-6 viremia as active HHV-6 infection with a causative role in pancytopenia and immune suppression. Foscarnet treatment resulted both in viral load decline and disappearance of HHV-6-related bone marrow suppression and predisposition to severe infections. Clinicians should be aware of the wide array of clinical manifestations and the diagnostic pitfalls of post-transplant HHV-6 disease. These issues are extremely challenging, as they may result either in dangerous underestimation of HHV-6 disease or in the institution of unnecessary antiviral therapy. Late bone marrow aplasia and late severe infections after allo-HCT without other obvious causes may be HHV-6 related.

Alloreactive microenvironment after human hematopoietic cell transplantation induces genomic alterations in epithelium through an ROS-mediated mechanism: in vivo and in vitro study and implications to secondary neoplasia.

We hypothesized that chronic tissue stress due to interaction of alloreactive donor cells with host epithelium after allogeneic hematopoietic cell transplantation (allo-HCT) may cause genomic alterations. We therefore analyzed 176 buccal samples obtained from 71 unselected allotransplanted patients for microsatellite instability (MSI). MSI was observed in 52% of allotransplanted patients but never in 31 healthy or autotransplanted controls. The patient age, the donor age, a female-to-male transplantation and a low number of CD34(+) cells in the graft were significantly correlated with genomic instability. There was a trend for increasing risk of MSI for patients who experienced severe graft-vs-host disease. Secondary malignancy was diagnosed in five (14%) of the MSI(+) and only in one (3%) MSI(-) patient. In an in vitro model of mutation analysis we found significant induction of frameshift mutations and DNA strand breaks in HaCaT keratinocytes co-cultured with mixed lymphocyte cultures (MLCs) but not after their exposure to interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta (TGF-beta), MLC supernatant, peripheral blood mononuclear cells (PBMCs) or phytohemagglutinin-stimulated PBMC. A reactive oxygen species-mediated mechanism is implicated. The in vivo and in vitro data of our study show that alloreactions after allo-HCT may induce genomic alterations in epithelium. Progress in understanding DNA damage and repair after allo-HCT can potentially provide molecular biomarkers and therapeutic targets.

Inherited thrombophilia screening in Greek women with recurrent fetal loss.

OBJECTIVE: The present study was designed to determine the prevalence of factor V Leiden (FVL), prothrombin gene G20210A (PTG) and methylenetetrahydrofolate reductase (MTHFR C677T) mutations in women from South-Western Greece with recurrent fetal loss (RFL) and negative personal thromboembolic history. MATERIALS AND METHODS: 212 women with RFL and 181 women with at least two pregnancies with normal outcome and no history of pregnancy loss were investigated for the commonest thrombophilic mutations (FVL, PTG, MTHFR C677T). Comparisons between groups were performed by Pearson's chi-square test and odd ratios were calculated. RESULTS: An abnormal genotype was detected in 49 women of the study group (23.1%) and in 41 women of the control group (22.6%). CONCLUSION: Inherited thrombophilia screening is not indicated as an initial approach in Greek women with RFL and negative personal thromboembolic history.

c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

Procleix Ultrio transcription-mediated amplification vs. serological blood screening in south-western Greece.

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38,264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1.4 per thousand), 8 for HCV (0.2 per thousand) and none for HIV or multiple infections. The yield of TMA was 0.183 per thousand for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.

Effect of hydroxyurea on the deformability of the red blood cell membrane in patients with sickle cell anemia.

Sickle cell disease is characterized by vaso-occlusive episodes, mainly in the small vessels, resulting in tissue ischemia, multi-organ failure, and, occasionally, death. Hydroxyurea (HU) is an agent with important and effective role in the treatment of patients suffering from this disease. The purpose of this study was to estimate the effect of HU on the deformability of the red blood cell's membrane (RBCM) in an effort to possibly improve the rheological properties of the RBCs of patients with sickle cell anemia (SCA), as well as to investigate the mechanical and rheological properties of these cells using micropipette and filtration techniques. The rigidity index, IR, which is a measure of cell rigidity and the elastic shear modulus, mu, which is a measure of cell's membrane deformability (CMd), of the RBCs from normal subjects, used as normal controls, were found significantly lower as compared to those of patients with SCA, regardless the treatment with HU. Patients under treatment with HU exhibited values better than those of untreated patients, in both, IR as well as mu, although still worse than the values of normal controls.

Constitutional pericentric inversion of chromosome 9 and hematopoietic recovery after allogeneic stem cell transplantation.

Recent reports suggest that hemopoietic stem cells with constitutional pericentric inversion of chromosome 9 [inv(9)] may be related to delayed engraftment or hemopoietic defect after stem cell transplantation (SCT). We conducted a retrospective study on five allogeneic SCT in which constitutional inv(9) was detected either in the donor or the recipient. The results showed that hematologic recovery was within the expected time range for all our patients. However, one patient exhibited decreasing blood counts between day +45 and +272 after transplantation, possibly due to protracted cytomegalovirus (CMV) infection and gansiclovir and imatinib treatment. Our findings suggest that constitutional inv(9) may not be associated with delayed hemopoietic recovery after SCT.

Dendritic cells in patients with type I Gaucher disease are decreased in number but functionally normal.

Gaucher disease is a lysosomal storage disorder, in which undigested glucosylceramide is deposited in the cytoplasm of mature macrophages, which accumulate in the bone marrow and the reticuloendothelial system. Dendritic cells are bone marrow-derived cells, specialized for the uptake, processing, transport and presentation of antigens to T-lymphocytes. We investigated peripheral blood dendritic cell-precursors, as well as the potential of peripheral blood monocytes and bone marrow-derived progenitor cells, to differentiate into mature dendritic cells in 12 patients with type I Gaucher disease. Results of the 10 adult patients were compared with those of 10 healthy volunteers, matched for age and sex. Six patients were anemic and 9 were thrombocytopenic, but none had severe bone disease. Both myeloid and plasmacytoid dendritic cells of patients with Gaucher disease, as well as the yield of the monocyte-derived dendritic cells, obtained after GM-CSF and IL-4 stimulation, were found significantly decreased, when compared to controls (myeloid dendritic cells: 0.19 +/- 0.07% vs. 0.34 +/- 0.10%, P = 0.009, plasmacytoid dendritic cells: 0.17 +/- 0.12% vs. 0.39 +/- 0.13%, P = 0.004, monocyte-derived dendritic cells: 4.8 +/- 3.5% vs. 8.3 +/- 3.2%, P = 0.036). However, the immunophenotypic profile of dendritic cells, estimated by CD1a, CD40, CD54, CD80, CD83 and HLA-DR expression, the endocytic and allo-stimulatory capacity of the immature, as well as of the TNF-alpha- or lipopolysaccharite-stimulated mature monocyte-derived dendritic cells, was similar to those obtained by healthy controls. In addition, bone marrow-derived CD34+ cells differentiated in the presence of GM-CSF, SCF, TNF-alpha and IL-4 into mature dendritic cells that did not differ in number, phenotype and allo-stimulatory activity from those of controls. Our findings suggest that patients with Gaucher disease exhibit mainly quantitative defects of their dendritic cells' system, demonstrated by decreased circulating dendritic cell precursors of both myeloid and plasmacytoid type. This finding may contribute to the poor immune response against infectious agents and an impaired immune surveillance, associated with an increased risk of developing a neoplastic disease.

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element.

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34(+) cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects.

Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

Virological, immunological and histological aspects in adult beta-thalassemic patients with chronic hepatitis C virus infection.

OBJECTIVES: Multitransfused adult beta-thalassemic patients constitute a population with high prevalence of hepatitis C virus (HCV) infection, because of transmission of HCV from infected blood donors prior to the introduction of anti-HCV screening. The aim of this study was to compare them with otherwise normal patients with HCV infection. METHODS: Forty-two adult multitransfused beta-thalassemics and 49 otherwise normal patients of the same age, with chronic HCV infection were studied. Viral parameters, autoimmunity indices and liver histology were evaluated. RESULTS: Serum HCV RNA levels were found significantly lower in thalassemic (median: 65,150 international units per milliliter (IU/ml); range: 3 059 380 IU/ml) than in non-thalassemic (NT) patients (median: 580,000 IU/ml; range: 10,956,000 IU/ml; P=0.001). The most prevalent genotype in thalassemic group was genotype 4 (32.4%) while in NT group was genotype 3a (59.2%). Cryoglobulins were detected in 8/42 (19%) thalassemic patients and in 12/49 (24.5%) NTs. Thalassemic patients had significantly lower levels of C3 and C4 components of complement and higher incidence of anti-nuclear antibodies than those without thalassemia. In patients with thalassemia a lower grading score was noted in liver biopsy compared with those without thalassemia (4.41+/-1.98 vs 5.38 +/- 2.09, P=0.038). On the contrary, thalassemic patients were found to have a higher staging score (3.08 +/- 1.51 vs 2.33 +/- 1.34, P=0.024). CONCLUSIONS: Adult beta-thalassemic patients, compared with other patients with HCV infection, present lower necroinflammatory activity and lower viral load but higher staging score. Autoimmune features are marginally different. Age of acquiring the infection, iron overload and modulation of immune system by transfusions are the proposed causes of these differences.

Determination of plasma cell secreting potential as an index of maturity of myelomatous cells and a strong prognostic factor.

According to the widely accepted myeloma staging system, the bulk of paraprotein is the main determinant of disease stage. However, myelomatous plasma cells differ considerably in their ability to synthesize and secrete monoclonal paraprotein. We determined plasma cell secreting potential (PCSP) as the amount of M-component, divided by the percentage of marrow plasmacytic infiltration, in 240 patients with myeloma, and correlated our results with chain isotype, plasma cell morphology, severity of bone disease, well-recognized prognostic factors, such as serum LDH, CRP, albumin and beta2-microglobulin, treatment response and overall survival. PCSP was higher in IgG than in other myeloma types, and was an almost constant parameter for each individual patient, in 134/166 cases. A > 10% decrease of PCSP in 26 patients was associated with disease aggressiveness and treatment failure. Patients with MGUS had significantly higher PCSP than those with myeloma of the same chain type. Higher PCSP was associated with stage I, absence of Bence-Jones proteinuria and indolent forms of disease with lower proliferating cell nuclear antigen (PCNA) positivity, serum LDH, alpha2-globulins, CRP and beta2-microglobulin and higher albumin levels. Conversely, patients with immature/plasmablastic morphology and those with severe bone disease had lower PCSP. Good responders to treatment had significantly higher PCSP than moderate and poor responders and PCSP was strongly correlated with overall survival in IgG and IgA myeloma. In conclusion, PCSP reflects the maturation status of myelomatous cells and therefore can be used as a prognostic factor, since patients with high secreting potential represent a lower malignancy group, in comparison to those with a low secreting potential.

Ticlopidine-induced aplastic anemia: two new case reports, review, and meta-analysis of 55 additional cases.

Ticlopidine-induced aplastic anemia (TIAA) is considered very uncommon. We present two new cases, and we review 55 additional cases from the literature. The first case concerns a 70-year-old man who developed severe aplastic anemia 7 weeks after treatment with 500 mg of ticlopidine daily. The patient sustained a severe septic episode, was treated with antibiotics and GM-CSF, and recovered the 14(th) day after ticlopidine withdrawal. The second was an 82-year-old man receiving ticlopidine for 2 years when, during a febrile episode, he was found neutropenic with marrow aplasia. Ticlopidine withdrawal and treatment with antibiotics, transfusions, and G-CSF helped him to recover. When the data of the 57 patients are evaluated, a reversible direct cytotoxic effect of ticlopidine on the pluripotent/bipotent hematopoietic progenitor stem cell is proposed. It is estimated that the real incidence if TIAA is higher, and many cases, initially presented as agranulocytosis +/- thrombocytopenia, might be true aplastic anemias, not proven by marrow aspiration or trephine biopsy. There is no effective monitoring to prevent this side effect. Recombinant growth factors appear not to help in shortening the neutropenic period.

Right ventricular cardiomyopathy in beta-thalassaemia major.

AIMS: To evaluate right ventricular function in patients with beta-thalassaemia major and congestive heart failure. Background In patients with beta-thalassaemia major a high incidence of cardiac involvement still exists despite improved prognosis with chelation therapy. Development of severe right heart failure is common and has been attributed to pulmonary hypertension secondary to lung haemochromatosis. However, the possibility of direct right ventricular myocardial involvement in the absence of significant pulmonary hypertension has not been adequately investigated. METHODS: Twenty-nine consecutively screened patients with beta-thalassaemia major and congestive heart failure were investigated by Doppler echocardiography, right ventricular first-pass radionuclide examination and cardiac catheterization. Haemodynamic data were obtained both before and after volume loading. A control group of 39 patients with beta-thalassaemia major, free from cardiac disease, and matched for age, gender, body surface area and heart rate was used for comparison. A subset of the control thalassaemic group (n=15) underwent both radionuclide and haemodynamic assessment. RESULTS: The majority of patients were on non-optimal chelation therapy. Only two of 29 patients were found to have cor pulmonale. One other patient suffered from constrictive pericarditis. A restrictive filling pattern in both ventricles and left ventricular systolic dysfunction were evident in the other 26 patients. Pulmonary artery pressure (systolic, 33+/-8 vs 27+/-5 mmHg, P<0.05) and pulmonary vascular resistance (114+/-56 vs 65+/-29 dynes. s. cm(-5), P<0.01) were only mildly elevated in the heart failure group. After volume challenge, cardiac output remained unchanged although the increments of ventricular filling pressures were significant (Deltaright atrial: 4.8+/-2.2 mmHg, P<0.05; Deltapulmonary capillary wedge: 5.6+/-2.9 mmHg, P<0.05) and correlated with each other (r=0.69;P<0.001) in heart failure patients, suggesting pericardial constraint and ventricular interaction. In these patients compared with the control thalassaemic group, a lower right ventricular ejection fraction (29%+/-9 vs 59%+/-6, P<0.0001) without correlation with pulmonary artery pressures was found. Haemodynamically significant right ventricular dysfunction defined as mean right atrial pressure >10 mmHg and ratio of mean right atrial-to-capillary wedge pressure >0.8 was evident in 15 of the 26 patients (58%), all with severe symptoms, representing three fourths of the patients in functional class III and IV. Simultaneous pressure recordings in six of these 15 patients showed equalization of ventricular end-diastolic pressures within 5 mmHg. CONCLUSION: The majority of patients with beta-thalassaemia major and severe congestive heart failure demonstrated a unique haemodynamic pattern similar to that described in predominant right ventricular infarction, indicating severe right ventricular cardiomyopathy in addition to left ventricular dysfunction. The incidence of cor pulmonale as a cause of right heart failure seems to be much lower than previously hypothesized.