Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

Quality of life and social integration after allogeneic hematopoietic SCT.

In total, 124 adult patients in remission after allogeneic hematopoietic SCT (HSCT) participated in a cross-sectional study to assess health-related quality of life (HRQL). Assessment of HRQL was carried out using two questionnaires: the (EORTC QLQ-C30) and the Functional Assessment of Cancer Therapy (FACT) with specific modules for BMT (FACT-BMT). Transplanted patients differed from healthy controls in many HRQL-related dimensions in the EORTC QLQ-C30: social functioning 73.4 versus 85.8, P<0.0001; role functioning 74.6 versus 83.3, P<0.004; physical functioning 83.9 versus 89.9, P<0.001; emotional functioning 72.2 versus 82.8, P<0.0001 but were not significant for global HRQL 71.2 versus 75.3, P<0.03. In total, 60% of the patients returned to work after HSCT; 31% part time and 29% full time. Age at HSCT and employment status were significantly associated with HRQL. Other factors such as disease and disease stage and especially the occurrence of late complications did not impact the perception of HRQL. This study suggests that the perception of HRQL after HSCT differs from the general population. Issues to increase work-related capabilities and improve social support need to be addressed.

Primary invasive aspergillosis of the digestive tract: report of two cases and review of the literature.

BACKGROUND: Disseminated aspergillosis is thought to occur as a result of vascular invasion from the lungs with subsequent bloodstream dissemination, and portals of entry other than sinuses and/or the respiratory tract remain speculative. METHODS: We report two cases of primary aspergillosis in the digestive tract and present a detailed review of eight of the 23 previously-published cases for which detailed data are available. RESULTS AND CONCLUSION: These ten cases presented with symptoms suggestive of typhlitis, with further peritonitis requiring laparotomy and small bowel segmental resection. All cases were characterized by the absence of pulmonary disease at the time of histologically-confirmed gastrointestinal involvement with vascular invasion by branched Aspergillus hyphae. These cases suggest that the digestive tract may represent a portal of entry for Aspergillus species in immunocompromised patients.

Haematopoietic stem cell transplantation in Switzerland. Report from the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) Registry 1997-2003.

In 1997, the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) initiated a mandatory national registry for all haematopoietic stem cell transplants (HSCT) in Switzerland. As of 2003, information was collected of 2010 patients with a first HSCT (577 allogeneic (29%) and 1433 autologous (71%) HSCT) and 616 additional re-transplants. This included 1167 male and 843 female patients with a median age of 42.4 years (range 0.2-76.6 years). Main indications were leukaemias (592; 29%) lymphoproliferative disorders (1,061; 53%), solid tumours (295; 15%) and non-malignant disorders (62; 3%). At the time of analysis 1,263 patients were alive (63%), 747 had died (37%). Probability of survival, transplant related mortality or relapse at 5 years was 52%, 21%, 36% for allogeneic and 54%, 5%, 60% for autologous HSCT. Outcome depended on indication, donor type, stem cell source and age of patient. HSCT is an established therapy in Switzerland. These data describe current practice and outcome.

Impact of high-resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland.

It is currently unknown what degree of human leukocyte antigen (HLA)-mismatching is acceptable in unrelated donor hematopoietic stem cell transplantation (UD-HSCT). Mismatches at some loci may be more permissive than others. We have analyzed the effect of high-resolution HLA-matching on outcome of all 214 consecutive recipients of UD-HSCT carried out in Switzerland. All typing was by the Swiss reference laboratory. Donor-recipient pairs were HLA-10/10 matched (n=130) or mismatched for either HLA-A/-B/-DRB1/multiple loci (n=33; (HLA-A/-B=10); (-DRB1=8); (multiple=15)); HLA-C (n=29) or HLA-DQ/-DRB3 (n=22; (DQ=16); (-DRB1=6)). The median follow-up was 32 months. Survival probabilities (+/-95% confidence interval) at 3 years were 57 (+/-10)% for recipients of HLA 10/10-matched transplants, 53 (+/-22)% for recipients of HLA-DQ/-DRB3-mismatched transplants, 44 (+/-20)% for recipients of HLA-C-mismatched transplants and 0% for recipients of transplants mismatched at HLA-A/-B/-DRB1/multiple loci (P<0.0001). In multivariate analyses, HLA compatibility was the variable most significantly associated with survival and treatment-related mortality. We found important differences in survival in recipients of UD-HSCT with best results for transplants from 10/10 matched donors. Single mismatches at HLA-DQ/-DRB3 were well tolerated, mismatches at HLA-C had intermediate results and mismatches at HLA-A/-B/-DRB1/multiple loci resulted in poor survival.

Stem cell transplantation from identical twins in patients with myelodysplastic syndromes.

In a multicentre retrospective EBMT database study, we analysed factors influencing outcome in 38 patients with MDS/sAML who were transplanted with stem cells from their syngeneic twin and compared those to 1444 patients who were transplanted from an HLA-identical sibling. The median time to leukocyte and platelet engraftment was faster in the twin group: 14 vs 17 (P=0.02) and 16 vs 26 days (P=0.09), respectively. The 5 years cumulative incidence of treatment-related mortality (TRM) was higher in the sibling than in the twin group (38 vs 27%; P=0.05). The 5 year cumulative incidence of relapse was 32% (95% CI: 29-35%) for the siblings and 39% (95% CI: 26-60%; P=0.6) for the twins. A trend for better 5-years disease-free and overall survival was observed in the twin group: 34% (95% CI: 14-54%) vs 28% (95% CI: 25-31%; P=0.2) and 36% (95% CI: 15-57%) vs 32% (95% CI: 29-35%; P=0.09), respectively. In a multivariate analysis, stem cell transplantation from identical twins had a lower TRM: HR: 0.4 (95% CI: 0.2-0.9; P=0.03). The relapse rate was similar for both groups with a HR of 1.2 (95% CI: 0.07-2.1; P=0.5), with a better survival for the twins: HR 0.6 (95% CI: 0.4-1.0; P=0.07). We conclude that twin transplantation in MDS/sAML is associated with a similar relapse risk, a lower TRM and a trend for better overall survival in comparison to transplantation from HLA-identical siblings.

Isolated HLA-C mismatches in unrelated donor transplantation for CML.

HLA-incompatibility is a major factor associated with outcome of allogeneic stem cell transplantation, but little is known on the impact of isolated HLA-C mismatches. We analyzed the outcome of 114 CML patients transplanted with marrow from unrelated donors of whom 24 were mismatched for HLA-C only (9/10 match). Univariate estimates of 5-year survival (SRV) (median follow-up: 47 months) in the HLA-matched group were 68+/-12 vs 42+/-20% (P=0.03) for the patients mismatched for HLA-C only and 33+/-33% in the mismatched group (non-HLA-C single mismatches and multiple mismatches) (P=0.0004). Disease stage, GVHD-prophylaxis (T-cell depletion), CMV-status and HLA-incompatibility were the risk factors associated (all P< or =0.005) with poor outcome. In the multivariate analysis, patients mismatched for loci other than HLA-C were at high risk of an adverse outcome (death: RR, 2.9; CI, 1.6-5.4, P=0.008, transplant-related mortality (TRM): RR, 3; CI, 1.5-5.9, P=0.0015). For patients mismatched for HLA-C only, the increased risk was of borderline significance (death: RR, 1.9; CI, 1-3.9, P=0.06, TRM: RR, 2.1; CI, 1-4.5, P=0.07). In spite of their lower expression, HLA-C antigens still represent relevant transplantation barriers that should be considered when searching for an unrelated donor.

Quality control of a national bone marrow donor registry: results of a pilot study and proposal for a standardized approach.

Unrelated hematopoietic stem cell transplantation (HSCT) is a recognized therapy for hematological diseases and over 8 million HLA-typed donors are ready to donate. Increased international exchanges and rapid requests through the Bone Marrow Donor Worldwide (BMDW) ask for standardized quality assurance. Since no such standards have been established to date, we tested a pilot program in order to evaluate donor availability and quality of HLA typing of the Swiss Registry. The 18500 donors of the registry have been analyzed by serology for HLA-AB and by molecular typing for HLA-DR. Through three successive annual quality control (QC) exercises, a total of 114 donor requests were sent to 13 blood transfusion centers responsible for donor recruitment asking for a blood sample. Donors were randomly selected according to recruitment periods (1988-1993; 1994-1997; 1998-2000), and to homozygosity for HLA-A and/or -B antigens. An additional 80 frozen blood samples from the repository corresponding to the three periods (n=26) and to the 2001 period (n=54) were also included in the HLA study. HLA-AB typings were done by polymerase chain reaction-sequence specific primers (PCR-SSP) and all discrepancies were retyped. The results showed that 79 samples provided by 69.3% of the requested donors were received within 14 days, and 19 samples (16.7%) were received in >14 days. Altogether, an 86% rate of donor availability was observed, independent of the recruitment period. Among the requested donors, 16 (14%) were not available: for medical reasons (two), for personal reasons (eight), for loss (one), and for an unknown reason (five). The HLA-A/B DNA typing results of 166 homozygous and 12 heterozygous blood samples showed that 437/439 (99.5%) of the assigned A/B antigens were correct. However in 36/178 donors (20.2%) an HLA-A or -B antigen had been missed (34 donors) or misassigned (two donors) by serology, with a decreasing discrepancy rate of 30% (1988-1993) to 18.5% in 2001. Assuming that HLA-A or -B homozygotes are found in 10-15% of the donors and that correct assignments have been observed in nearly 100% of the donors, an overall error rate of 4-5% would be expected for the national registry HLA-AB typing. These data show that standardized quality control for donor availability and HLA typing is feasible, and we propose that this model could be applied to the registries participating in bone marrow donor worldwide.

Optimized lentiviral transduction of erythroid precursors from healthy adults and patients with myelodysplastic syndromes.

Lentivectors, derived from human immunodeficiency virus-1 (HIV-1), represent a novel investigational and therapeutic tool for targeting hematopoietic progenitor cells. We describe a new protocol whereby we achieved a highly efficient lentiviral transduction of erythroid precursor cells originating from the bone marrow of healthy adults and patients with myelodysplastic syndromes (MDS). CD34(+) stem cells from healthy subjects were cultured with erythropoietin, IL-3 and stem cell factor, and thereby expanded approximately 300-fold. When these cultures were transduced with a lentiviral vector expressing GFP as a reporter gene, 70% glycophorin(+) cells were GFP(+). Although proliferation and levels of transduction were reduced in cultures of CD34(+) stem cells from patients with myelodysplastic syndromes, 50% of glycophorin(+) cells became GFP(+), amongst which 30% were sideroblastic erythroid precursors. This study demonstrates that lentiviral vectors are capable of efficiently transducing MDS precursors and offers new perspectives to investigate the influence of specific genes on normal erythroid differentiation. This may eventually help to correct defects in patients suffering from myelodysplastic syndromes.

Frequency response of renal sympathetic nervous activity to aortic depressor nerve stimulation in the anaesthetized rat.

1. The contribution of central baroreceptor reflex pathways to the dynamic regulation of sympathetic nervous activity (SNA) has not been properly examined thus far. The aim of this study was to characterize the transfer function of the central arc of the baroreceptor reflex (from baroreceptor afferent activity to SNA) over a wide range of frequencies. 2. In nine baroreceptor-intact and six sino-aortic baroreceptor-denervated rats anaesthetized with urethane, the renal SNA was recorded while applying sinusoidal stimulation to the aortic depressor nerve at 26 discrete frequencies ranging from 0.03 to 20 Hz. At each modulation frequency, cross-power spectrum analysis using a fast Fourier transform algorithm was performed between the stimulation and renal SNA, which provided the transfer function of the central arc. 3. In both baroreceptor intact and denervated rats, the transfer gain increased by a factor of about three between 0.03 and 1 Hz. At higher frequencies, the gain decreased but remained above the static gain of the system up to 12 Hz. There was a slight phase lead up to 0.4 Hz, then a continuously increasing phase lag. A three-element linear model satisfactorily described the experimental transfer function. The model combined a derivative gain (corner frequency approximately 0.15 Hz), an overdamped second-order low-pass filter (natural frequency approximately 1 Hz) and a fixed time delay (approximately 100 ms). 4. These results indicate that the central arc of the baroreceptor reflex shows derivative properties that are essential for compensating the filtering of fast oscillations of baroreceptor afferent activity and thus for the generation of fast oscillations of renal SNA (e.g. those related to the cardiac cycle).

Allogeneic bone marrow transplantation is associated with a preferential femoral neck bone loss.

Osteoporosis is a major complication of organ transplantation. Little is known about the risk of developing osteoporosis in bone marrow transplant (BMT) recipients. We studied early and late changes in bone mineral density (BMD), as well as biochemical markers of bone remodeling, in patients at the time of allogeneic BMT (alloBMT) and up to 13 years thereafter. In a cross-sectional study, 102 patients (40 women, 62 men, mean age +/- SEM, 38.9 +/- 1.6 years) were segregated into a first group (A, n = 48) and evaluated before or during the first weeks (mean +/- SD 0.3 +/- 0.1 month, range -0.5 to 3 months) following alloBMT, and a second group (B, n = 54) studied 60.1 +/- 5.6 months (range 6-156 months) following alloBMT. Lumbar spine (LS) BMD was similar in groups A and B and was within normal limits. In contrast, femoral neck (FN) Z- and T-scores were significantly decreased in group B compared with group A (-0.68 +/- 0.14 vs -0.03 +/- 0.14 SD and -0.84 +/- 0.14 vs -0.22 +/- 0.14 SD, respectively; p < or = 0.002). Osteopenia (T-score between -1 and -2.5 SD) was present in 35% of group A and 43% of group B patients (NS). Osteoporosis (T-score < -2.5 SD) was detected in 7% of group B patients, but in none of those in group A (p = 0.05). In a longitudinal study, 56 subjects were evaluated at the time of alloBMT, and 33 and 23 were studied 6 or 12 months later, respectively (13 women, 20 men, 37.5 +/- 1.6 years). All were treated with supplements of calcium and vitamin D. Amenorrheic women received hormone replacement therapy (HRT). Three-monthly pamidronate infusions were given to 15 men and 10 non-amenorrheic women who were osteopenic/osteoporotic or had elevated baseline bone turnover markers. Mean baseline LS and FN Z- and T-scores were within normal range. Six months after BMT, FN BMD decreased by 4.2 +/- 0.7% (p < 0.001), and whole body BMD and bone mineral content by 1.5 +/- 0.4% and 3.1 +/- 0.6%, respectively (p < or = 0.0001). Twelve months after the graft, there was no further significant bone loss and only FN BMD decrease remained significantly different compared with baseline (-5.6 +/- 1.1%, p < or = 0.0001). These results indicate that the risk of decreased BMD is higher for the femoral neck than the lumbar spine and whole body levels in patients with allogeneic bone marrow transplantation, and that bone loss occurs mainly during the first 6 months after the graft.

Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells.

BACKGROUND: Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this reason, the production of transgene-expressing DC derived from CD34 + cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated. METHODS: CD34+ cells were transduced with lentivectors encoding for GFP before or after 21 days of culture with FLT3-ligand, thrombopoietin and stem cell factor and induction into DC with GM-CSF+IL-4 (G4) or G4+TNF (GT4). GFP and DC-specific marker expression was assessed by flow cytometry, and allostimulatory capacity was evaluated on GFP+ and GFP- sorted cells. RESULTS: Immature (G4-induced) DC obtained from amplified CD34 + cells were transducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large quantities of vectors were required to generate transgene-expressing cells with this protocol. In contrast, greater numbers of both immature and mature GFP- expressing DC were obtained with CD34+ cells exposed to lentivector shortly after purification. By the time of DC induction, GFP+ cells had increased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, HLA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+GFP+ DC that displayed a higher allostimulatory capacity than cells induced with G4 alone. CONCLUSION: The transduction of a small number of CD34+ cells with minimal doses of lentivector may allow for the production of a large number of DC expressing selected antigens useful for immunotherapy.

CD34(+) cord blood cells expressing cutaneous lymphocyte-associated antigen are enriched in granulocyte-macrophage progenitors and support extensive amplification of dendritic cell progenitors.

OBJECTIVE: We evaluated the frequency of hematopoietic progenitor cells (HPC) in CD34(+)CLA(+) (cutaneous lymphocyte-associated antigen) and CD34(+)CLA(-) cord blood cells, and followed cellular growth and HPC production during cultures in Flt3 ligand, thrombopoietin, and stem cell factor (FTS). MATERIALS AND METHODS: Immunomagnetic bead-purified CD34(+) cells were sorted into CD34(+)CLA(+) or CD34(+)CLA(-) cells. HPC frequency was assessed by clonal assays in methylcellulose either ex vivo or after, 7, 14, or 21 days of culture with FTS. Dendritic cell (DC) progenitors were evaluated after induction of FTS-amplified cells into DC using secondary cultures containing granulocyte-macrophage colony-stimulating factor and interleukin-4. RESULTS: Ex vivo, granulocyte-macrophage progenitors were more frequent and erythroid progenitors were less frequent in the CLA(+) fraction. In FTS culture, CD34(+)CLA(+) cells produced greater absolute numbers of CD34(+) cells, granulocyte-macrophage-, erythroid-, and DC (including Langerhans cell-related) progenitors compared to CD34(+)CLA(-) cells. In CD34(+)CLA(+) cultures, CLA(+) cells steadily decreased with time, and CD34(+)CLA(-) cells appeared. In CD34(+)CLA(-) cultures, CLA(+) cells were generated, increased up to day 7, and decreased thereafter. CLA was expressed only on CD34(-) cells in these cultures. Ex vivo, CD34(+)CLA(+) cells could be subdivided further into CD38(low) and CD38(high) cells. Cord blood and growth factor-mobilized CD34(+) cells contained more CLA(+)CD38(low) cells than nonmobilized peripheral blood CD34(+) cells and proliferated more extensively with FTS than the latter cells. CONCLUSIONS: CD34(+)CLA(+) cells contain a rather immature progenitor capable of high proliferation and extensive amplification of HPC in vitro. This progenitor may be localized in the CD34(+)CLA(+)CD38(low) fraction. In addition, cultures of CD34(+)CLA(+) cells from cord blood produced CD34(+)CLA(-) cells, suggesting that these cells may derive directly from CD34(+)CLA(+) cells in vivo.

Effects of aerobatics flight on oxygen consumption and heart rate control: influence on autonomic cardiovascular regulation during recovery.

Oxygen consumption (VO2) and blood pressure regulation were measured on five pilots during and after normal training aerobatics flights of a mean duration of 35 min. The acceleration vector along the longitudinal axis of the body (Gz) ranged from + 6.5 Gz to -3.5 Gz. VO2 was continuously monitored by a miniature telemetric system (K2). Heart rate (fc), the abdominal muscle electromyogram (EMG) and Gz levels were recorded synchronously on a magnetic tape recorder. A tilt test was performed pre- and post-flight to evaluate fc and blood-pressure variability. The left forearm blood flow was measured by strain-gauge plethysmography. The mean VO2 during flight was 1.2 l x min(-1), with a peak VO2 of 2.1 l x min x fc ranged between 55 and 165 beats x min(-1) and showed a progressive increase under the effect of + Gz, with a sudden fall during -Gz. The abdominal muscle EMG indicated the occurrence of muscle contraction under Gz load. Maximal responses were observed during the Gz phase. Comparison between pre- and post-flight data showed lower post-flight systolic blood pressure with higher fc. Before flight, upright tilt induced a significant increase in low/ high frequency fc, as assessed using spectral analysis. This change was suppressed after flight. In summary, these data show that aerobatics flight leads to enhanced energy expenditure, mainly because of increased skeletal muscle work. The post-flight tilt test showed that aerobatic flight favors parasympathetic drive and, consequently, modifies blood pressure regulation during recovery. This action may decrease + Gz tolerance to a second aerobatics flight performed shortly after the first.

Human memory T cells: lessons from stem cell transplantation.

Animal models have revealed the rules for the organization of mature T-cell pools. However, in humans, little is known about memory T cells, which differ in lifespan and in the number of times that the same antigen is encountered. Here, Nathalie Rufer and colleagues discuss their findings in stem-cell-transplanted patients, which provide interesting data on the human T-cell compartment.

Total body irradiation before allogeneic bone marrow transplantation: is more dose better?

PURPOSE: This study was performed to retrospectively assess the potential influence of total-body irradiation (TBI) dose on overall survival in patients undergoing allogeneic bone-marrow transplants (BMT) for hematologic malignancies. METHODS AND MATERIALS: Between October 1984 and December 1996, 116 patients were conditioned with high-dose chemotherapy and fractionated TBI before allogeneic BMT. The median age was 34 years (range 3-60). The TBI dose was given in 6 fractions, twice-a-day, over 3 days before BMT. The total dose was 10 Gy in 24 patients, 12 Gy in 66 patients, and 13.5 Gy in 26 patients. RESULTS: TBI dose was inversely correlated with overall survival. Five-year survival was 62% for patients conditioned with 10 Gy, 55% for patients conditioned with 12 Gy, and 46% for patients conditioned with 13.5 Gy. Age at BMT was also independently correlated with survival, with the best outcome for patients < 40 years old. CONCLUSION: A TBI dose (fractionated) > 10 Gy may not necessarily be associated with a better outcome in patients undergoing allogeneic bone-marrow transplant for hematologic malignancies.

Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation.

Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.

Fatal myocardial aspergillosis in an immunosuppressed child.

A girl with resistant acute myeloid leukemia (AML) had a stem cell transplantation. Preceding transplantation, she had recurrent pneumonitis. No causative agent was identified. Despite several antibiotics including high-dose liposomal amphotericin-B, pulmonary infection progressed. Aspergillosis, always considered, could not be documented. She died from cardiac arrest on the second day after transplantation, with no forewarning of previous heart disease. Pericardial and myocardial aspergillosis was an autopsy finding. Pericardial and myocardial aspergillosis, rare manifestations of systemic aspergillosis, should be considered in any immunocompromised patient with long-lasting pulmonary infection, even in the absence of specific cardiac findings.

High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus-based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1alpha and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1alpha promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1alpha promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3' long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.