ALBERT

Description: Key Points Single-photon emission computed tomography imaging can be used to image immune recovery in lymphoid tissues following transplant. There is discordance between lymphoid tissues and the peripheral blood in numbers of CD4+ cells following various doses of irradiation.

Description: Murine erythroid progenitors infected with the anemia-inducing strain of Friend virus (FVA cells) undergo apoptosis when deprived of erythropoietin (EPO). When cultured with EPO, they survive and complete terminal differentiation. Although cell volume is decreased and nuclear chromatin is condensed during both apoptosis and terminal differentiation, morphologic and biochemical distinctions between these two processes were observed. In apoptosis, homogeneous nuclear condensation with nuclear envelope loss occurred in cells that had not reached the stage of hemoglobin synthesis. In terminal erythroid differentiation, nuclear condensation with heterochromatin, euchromatin, and nuclear envelope preservation occurred simultaneously with hemoglobin synthesis. Cells with apoptotic morphology appeared asynchronously in EPO-deprived cultures, indicating that only a portion of the cells were undergoing apoptosis at any given time. The percentages of apoptotic cells and cleaved DNA increased with time in EPO-deprived cultures. Inhibition of DNA cleavage was directly proportional to EPO concentration over a wide physiologic range, demonstrating a heterogeneity in susceptibility to apoptosis based on variability in the EPO sensitivity of individual cells. A subpopulation of FVA cells with increased EPO sensitivity (decreased EPO requirement) was isolated from EPO-deprived cultures. This increased EPO sensitivity did not result from differences in EPO receptor number, affinity, or structure, suggesting that the differences are in the signal transduction pathway. These results indicate that control of red blood cell production involves both prevention of apoptosis by EPO and heterogeneity in the EPO requirement of individual progenitor cells.

Description: Murine erythroid progenitors infected with the anemia-inducing strain of Friend virus (FVA cells) undergo apoptosis when deprived of erythropoietin (EPO). When cultured with EPO, they survive and complete terminal differentiation. Although cell volume is decreased and nuclear chromatin is condensed during both apoptosis and terminal differentiation, morphologic and biochemical distinctions between these two processes were observed. In apoptosis, homogeneous nuclear condensation with nuclear envelope loss occurred in cells that had not reached the stage of hemoglobin synthesis. In terminal erythroid differentiation, nuclear condensation with heterochromatin, euchromatin, and nuclear envelope preservation occurred simultaneously with hemoglobin synthesis. Cells with apoptotic morphology appeared asynchronously in EPO-deprived cultures, indicating that only a portion of the cells were undergoing apoptosis at any given time. The percentages of apoptotic cells and cleaved DNA increased with time in EPO-deprived cultures. Inhibition of DNA cleavage was directly proportional to EPO concentration over a wide physiologic range, demonstrating a heterogeneity in susceptibility to apoptosis based on variability in the EPO sensitivity of individual cells. A subpopulation of FVA cells with increased EPO sensitivity (decreased EPO requirement) was isolated from EPO-deprived cultures. This increased EPO sensitivity did not result from differences in EPO receptor number, affinity, or structure, suggesting that the differences are in the signal transduction pathway. These results indicate that control of red blood cell production involves both prevention of apoptosis by EPO and heterogeneity in the EPO requirement of individual progenitor cells.

Description: We examined the synthesis of lactoferrin, an iron binding protein that, among hematopoietic cells, is restricted to secondary granules of polymorphonuclear leukocytes. Lactoferrin biosynthesis was absent from leukemic myeloblasts and promyelocytes but abundant in normal bone marrow and both the bone marrow and peripheral blood of patients with chronic myelogenous leukemia (CGL) if the samples contained substantial numbers of myelocytes and metamyelocytes. Lactoferrin was present in the steady state in normal or CGL bands and polymorphonuclear leukocytes, but no lactoferrin biosynthesis was detectable in these samples. Taken together, these results suggest that lactoferrin accumulation begins with the onset of biosynthesis at the myelocyte stage and is largely complete by the beginning of the band stage of maturation. HL-60 cells, a permanent promyelocytic leukemia cell line, synthesized no lactoferrin. Translation of messenger RNA in Xenopus laevis oocytes revealed that mRNA from patients with chronic myelogenous leukemia and abundant myelocytes and metamyelocytes directed the synthesis of readily detectable amounts of lactoferrin, whereas HL-60 cells contained no translatable lactoferrin mRNA. We thus hypothesize that lactoferrin is a useful marker of gene expression restricted to the terminal stages of granulocyte maturation. Biosynthesis of this protein appears to be mediated by appearance of translatable mRNA at the myelocyte stage, coincident with development of secondary granules. Absence of lactoferrin production by HL-60 cells is due to absence of translatable lactoferrin mRNA, either because of lineage infidelity of these transformed cells or because of arrest before the developmental stage at which secondary granules appear.

Description: The susceptibility of highly purified human CD34+ cells to monocytotropic (Ba-L) and lymphotropic (A018-post) strains of human immunodeficiency virus-1 (HIV-1) was examined. Liquid cultures initiated with fresh immunomagnetically purified CD34+ cells using the K6.1 CD34 monoclonal antibody (MoAb) (K6.1/CD34+) were positive for HIV expression 2 weeks after exposure to HIV-1 Ba-L. These cells were initially greater than 90% CD34+ and had undetectable monocyte contamination by flow-cytometric staining and side-scatter analyses, respectively, and undetectable T-cell contamination by CD3 polymerase chain reaction (PCR) analysis. However, secondary CD34+ liquid cultures reselected from the primary liquid cultures 24 hours after HIV exposure by panning with the ICH3 CD34 MoAb (ICH3/CD34+) and maintained for an additional 14 days were negative for HIV expression. The ICH3-unbound cells were positive for both spliced and unspliced HIV RNA when exposed to HIV-1 Ba-L, and were DNA PCR positive when exposed to either monocytotropic or lymphotropic HIV-1. To further test that CD34+ cells were not infectible by HIV-1, we exposed K6.1/CD34+ cells continuously to HIV-1 in a culture system capable of maintaining and expanding primitive CD34+ cells. HIV-exposed K6.1/CD34+ cells proliferated and expanded as efficiently as uninfected cultures. However, when reselected magnetically using the K6.1 CD34 MoAb after expansion for 7 days, bound K6.1/CD34+ cells were again negative for HIV-1 expression, whereas unbound cells were positive for HIV-1 expression. These findings suggest that a sequential CD34+ cell-selection process, in which the two selections are separated by a brief culture period, can yield a population of CD34+ cells that are not infected with HIV-1. This process may be useful in the design of stem or progenitor cell- based transplantation therapies for HIV infection.

Description: Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P

Description: Objectives: Emergency room (ER) evaluation may differ when physicians see patients in the context of clinical trials compared to routine care. We aimed to determine whether patterns in the use of a systemic pretest probability (PTP) stratification tool and D-dimer testing change over time and whether this influences their clinical utility. Methods: Charts were reviewed of 974 cases that had D-dimer testing for VTE exclusion in two time periods; 474 consecutive patients evaluated between 07/02 and 10/02 and 500 between 07/04 and 10/04. The former cohort was managed by ER physicians that had just participated in a clinical trial on VTE diagnosis including PTP assessment and D-dimer testing, whereas the latter group had received no formal training after 2002. In both cohorts, pretest scoring as low, intermediate or high risk according to Wells criteria for DVT and PE was performed in every case since this was requested from the laboratory. D-dimer testing (Vidas® D-Dimer, Biomerieux) was performed only in the low and moderate risk group after reception of the PTP assessment form. Physicians were also asked to check out a form detailing individual Wells criteria leading to the overall assessment but were not mandated to do so in order to obtain the D-dimer result from the laboratory. Results: Pretest probability was evaluated as low, moderate or high in 66,7%, 31,9% and 1,3% vs. 78,4%, 21,2% and 0% (all comparisons are 2002 vs. 2004). There was a significant increase in proportion of undetailed evaluations of PTP (32,7% vs. 61%). Detailed forms in both cohorts had similar risk distribution (low/moderate risk 51,7/46,4% vs. 59,2/40,8%) whereas undetailed forms were usually quoted as low risk (97,4% vs. 90.5%). Number of D-dimer tests performed per month was stable over the three year period of observation and in the two evaluated cohorts (119/mth vs. 125/mth). D-dimer results were negative in 299/474 cases (63,1%) in 2002 and 359/500 (71,8%) in 2004 (p=0,003), although this difference was less apparent when analysed according to the individual PTP risk groups (low/moderate risk 77,3/51,9% vs. 71,2/48,3%). Incidence of VTE events decreased over time from 5,3 to 1,6% (p=0,002). Incidence in the low/moderate risk groups was 1,6%/10,6% vs. 0,3%/6,6%. Only one false negative result (popliteal vein DVT) was observed in the two cohorts (NPP = 99,9%). Conclusion: Our results show a decreasing incidence in VTE over time in an ER population screened by D-dimer testing and PTP even though the number of tests performed were stable over time. This was accompanied by a decreasing number of cases considered to have an intermediate PTP. These findings suggest a change of practice over time resulting in an increasing use of D-dimer testing for very low risk patients and a decrease in their use for intermediate risk patients. A decrease in the proper use of the PTP tool over time might result in overestimation of the physician perceived risk and therefore lead to an increase in imaging resource utilisation. Broader studies including imaging prescription trends over time will be needed to confirm this hypothesis.

Description: We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst- forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.

Description: Aspergillus fumigatus (AF) is a ubiquitous mold and is the most common cause of invasive aspergillosis, an important source of morbidity and mortality in immunocompromised hosts. Using cytokine flow cytometry, we assessed the magnitude of functional CD4+ and CD8+ T-cell responses following stimulation with Aspergillus antigens. Relative to those seen with cytomegalovirus (CMV) or superantigen stimulation, responses to Aspergillus antigens were near background levels. Subsequently, we confirmed that gliotoxin, the most abundant mycotoxin produced by AF, was able to suppress functional T-cell responses following CMV or staphylococcal enterotoxin B (SEB) stimulation. Additional studies demonstrated that crude AF filtrates and purified gliotoxin inhibited antigen-presenting cell function and induced the preferential death of monocytes, leading to a marked decrease in the monocyte-lymphocyte ratio. Analysis of caspase-3 activation confirmed that gliotoxin preferentially induced apoptosis of monocytes; similar effects were observed in CD83+ monocyte-derived dendritic cells. Importantly, the physiologic effects of gliotoxin in vitro were observed below concentrations recently observed in the serum of patients with invasive aspergillosis. These studies suggest that the production of gliotoxin by AF may constitute an important immunoevasive mechanism that is mediated by direct effects on antigen-presenting cells and both direct and indirect effects on T cells.

Description: The ability of a tumor cell to survive is critical for successful dissemination to sites distant from the primary tumor. Tumor cells must enter blood circulation, resist hemodynamic shear stress of the blood circulation, successfully extravasate, and then migrate through dense tissue stroma to a site favorable for tumor growth. Some tumor cells must therefore be endowed with peculiar abilities to successfully metastasize, whereas others, although capable of forming tumor in specific organs, cannot metastasize. This property has often been associated with the homing ability of a given tumor cell, likely through the expression of organ-specific homing receptors that are critical for the extravasation process. The present work was aimed at establishing the point at which metastatic and nonmetastatic lymphoma cells diverge. Although 164T2 and 267T2 lymphoma cell lines can successfully form thymic lymphoma when injected intrathymically, only the 164T2 clone can efficiently form tumor in kidneys, spleen, and liver after intravenous inoculation. Using the Indium-labeling technique to monitor the homing kinetic of both cell lines, we showed that the critical step for the successful metastasis of the lymphoma cell was determined in the final steps of the disseminating process, namely after homing. These results indicate that, whereas binding of tumor cells to vascular endothelium through specific adhesion mechanisms is a prerequisite for dissemination of tumor cells, the resistance of a tumor cell to the antagonist action of the host and/or its ability to grow tumor occurs only after homing to the target organ.