Circulating CD133<Superscript>+</Superscript>VEGFR2<Superscript>+</Superscript> and CD34<Superscript>+</Superscript>VEGFR2<Superscript>+</Superscript> cells and arterial function in patients with beta-thalassaemia major

Arterial dysfunction has been documented in patients with beta-thalassaemia major. This study aimed to determine the quantity and proliferative capacity of circulating CD133⁺VEGFR2⁺ and CD34⁺VEGFR2⁺ cells in patients with beta-thalassaemia major and those after haematopoietic stem cell transplantation (HSCT), and their relationships with arterial function. Brachial arterial flow-mediated dilation (FMD), carotid arterial stiffness, the quantity of these circulating cells and their number of colony-forming units (CFUs) were determined in 17 transfusion-dependent thalassaemia patients, 14 patients after HSCT and 11 controls. Compared with controls, both patient groups had significantly lower FMD and greater arterial stiffness. Despite having increased CD133⁺VEGFR2⁺ and CD34⁺VEGFR2⁺ cells, transfusion-dependent patients had significantly reduced CFUs compared with controls (p = 0.002). There was a trend of increasing CFUs across the three groups with decreasing iron load (p = 0.011). The CFUs correlated with brachial FMD (p = 0.029) and arterial stiffness (p = 0.02), but not with serum ferritin level. Multiple linear regression showed that CFU was a significant determinant of FMD (p = 0.043) and arterial stiffness (p = 0.02) after adjustment of age, sex, body mass index, blood pressure and serum ferritin level. In conclusion, arterial dysfunction found in patients with beta-thalassaemia major before and after HSCT may be related to impaired proliferation of CD133⁺VEGFR2⁺ and CD34⁺VEGFR2⁺ cells.     

CXCR4 expression on transplanted peripheral blood CD34<Superscript>+</Superscript> cells: relationship to engraftment after autologous transplantation in a cohort of multiple myeloma patients

Expression of the chemokine receptor CXCR4 by haematopoietic stem cells (HSCs) is believed to influence the process of these cells ‘homing’ back to the bone marrow post-transplantation, in response to the stromal cell-derived factor-1 gradient, followed by engraftment. The primary aim of this retrospective study was to compare reinfused CD34⁺ cell dose, assessed from the fresh collection, with the post-thaw viable (v) CD34⁺ and vCD34/CXCR4⁺ dual positive cell dose as predictors of haematopoietic recovery in multiple myeloma patients undergoing autologous stem cell transplantation. Cryopreserved samples from stem cell collections of 27 myeloma patients were analysed for CD34 and CXCR4 expression and times to haematological engraftment measured. Dosage of transplanted vCD34⁺ cells was on average 79% of the original calculation from the fresh collection bag (range 29–98%). The median percentage of vCD34+ cells co-expressing CXCR4 was 37% (3.7–97%). Surface expression of CXCR4 by thawed vCD34⁺ cells was closely correlated to complementary DNA levels. The median dose of CD34/CXCR4⁺ cells in the autografts was 1.2 × 10⁶/kg (0.2–3.0 × 10⁶/kg) compared with 3.3 × 10⁶/kg for transplanted vCD34⁺ cells (1.2–5.5 × 10⁶/kg). Both CD34 and vCD34 doses correlated with neutrophil engraftment (p < 0.005) although vCD34/CXCR4⁺ dose did not. However, patients given a higher dose of CD34/CXCR4⁺ cells (≥1.75 × 10⁶/kg) showed a faster time to platelet recovery (p < 0.05) than those given a lower dose (≤0.42 × 10⁶/kg). These results warrant further study of CD34/CXCR4 expression by mobilised HSCs and the relationship to platelet recovery post-transplantation on a larger cohort of patients.     

Single-dose pegfilgrastim is comparable to daily filgrastim in mobilizing peripheral blood stem cells: a case-matched study in patients with lymphoproliferative malignancies

Pegfilgrastim (PEGFIL) has been found to be comparable to daily filgrastim (FIL) in managing chemotherapy-induced neutropenia. In the present study, we evaluated the ability of PEGFIL to mobilize stem cells in 38 consecutive patients with lymphoproliferative diseases (multiple myeloma, n = 18; lymphomas, n = 15; chronic lymphocytic leukemia, n = 5). Patients were mobilized using PEGFIL (6–18 mg as a single dose) during 2005–2006; 32 then received high-dose chemotherapy followed by autologous stem cell transplantation. PEGFIL-mobilized patients were matched by age, disease, and treatment line at a ratio of 1:2 to historical FIL-mobilized controls. The primary study endpoint was the blood CD34⁺ concentration at onset of leukapheresis. Leukapheresis began a median of 10 days from the beginning of mobilization chemotherapy in both groups. At the onset of leukapheresis, median blood CD34⁺ cell counts did not differ significantly in the FIL group compared with the PEGFIL group (79 × 10⁶/L vs 64 × 10⁶/L, respectively; p = 0.44). In the different disease categories, the respective CD34⁺ cell counts after FIL and PEGFIL mobilization were 72 × 10⁶/L vs 123 × 10⁶/L (p = 0.08) in myeloma, 51 × 10⁶/L vs 62 × 10⁶/L (p = 0.6) in lymphomas, and 27 × 10⁶/L vs 30 × 10⁶/L (p = 0.62) in CLL, respectively. The target CD34⁺ cell yield was harvested with one leukapheresis in 53% of PEGFIL-mobilized patients. Engraftment after autografting did not differ significantly in the two groups. Stem cell mobilization with a single dose of PEGFIL was, therefore, comparable to that achieved using daily FIL in patients with lymphoproliferative diseases. PEGFIL is a more practical way to mobilize stem cells than daily FIL.     

Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34+ cord blood and bone marrow cells

 Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week long-term culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34⁺ BM and cord blood (CB) cells in 5-week LTCs. CD34⁺ cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34⁺ cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p<0.05). For CD34⁺ BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E output (p<0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34⁺ CB cells (p=0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-M1p, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for α-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34⁺ CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed. Received: February 16, 1999 / Accepted: June 21, 1999     

Factors influencing G-CSF-mediated mobilization of hematopoietic progenitor cells during steady-state hematopoiesis in patients with malignant lymphoma and multiple myeloma

 We retrospectively analyzed factors influencing PBPC mobilization during steady-state hematopoiesis in 52 patients with malignant lymphoma (n=35) or multiple myeloma (n=17) who received 77 cycles of G-CSF (12.5–50 μg G-CSF/kg/day). For 15 of these patients, the first mobilization cycle (12.5 μg G-CSF/kg/day) was followed by a second course with an increased dose of G-CSF (25 or 50 μg/kg/day). Leukapheresis was started on day 4, about 2 h after s.c. G-CSF administration, and repeated on 2–5 consecutive days. CD34+ cells were determined by flow cytometry in each apheresis product and in the peripheral blood prior to G-CSF administration, beginning on day 4. Colony assays were performed on cryopreserved samples prior to autografting. In the 15 patients receiving two mobilization cycles the higher G-CSF dose was associated with higher levels of CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful (>2×10⁶ CD34+ cells/kg) collections (p=0.058). Patients with limited previous cytotoxic therapy (n=19, up to six cycles of a standard regimen such as CHOP and/or less than 20% marrow irradiation) who received a daily dose of 12.5 μg G-CSF/kg had higher levels of circulating CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful collections (p<0.05) compared with patients previously treated with more intensive radiochemotherapy (n=15). Ten of 20 patients (50%) who failed during the first cycle were successful during subsequent cycles with escalated doses of G-CSF. Trough levels of circulating CD34+ cells on day 4 were predictive for success or failure to achieve >2×10⁶ CD34+ cells/kg, especially in heavily pretreated patients. In conclusion, a daily dose of 12.5 μg G-CSF/kg seems sufficient to mobilize PBPC during steady-state hematopoiesis in the majority of patients who have received limited previous radiochemotherapy. Higher doses of G-CSF, up to 50 μg/kg/day, mobilize more PBPC and should be considered for patients previously treated with intensive radiochemotherapy or those failing to mobilize sufficient numbers of CD34+ cells with lower doses of G-CSF. Received: December 15, 1998 / Accepted: April 28, 1999     

Production of matrix metalloproteinase-9 by cord blood CD34<Superscript>+</Superscript> cells and its role in migration

The transmigration of hematopoietic progenitor cells is a crucial step in the homing of transplanted stem cells into bone marrow (BM) microenvironment; however, the molecular basis for this is not fully understood. Matrix metalloproteinases (MMPs), which are implicated in the migration of leukocytes, are important in degrading components of extracellular matrix molecules. In this study, using zymographic analysis and enzyme-linked immunosorbent assay (ELISA), we investigated the production of MMP-9 in CD34⁺ cells from cord blood (CB) and BM, compared their spontaneous migration across a reconstituted basement membrane-coated filter in transwell, and studied the role of MMP-9 in the transmigration. Zymography and ELISA showed that MMP-9 is produced by freshly isolated CD34⁺ stem/progenitor cells obtained from CB. CB CD34⁺ cells showed significantly higher migrational capacity than BM CD34⁺ cells (p=0.008). Furthermore, the migrational ability of CB CD34⁺ cells over the extracellular matrix (ECM) was significantly inhibited by the inhibitor of MMP, o-phenanthroline and anti-MMP-9 monoclonal antibody (73.3±11.8% and 37.5±10.4% inhibition, respectively). Our results strengthen the potential role of MMP-9 in the higher migrational capacity of CB CD34⁺ cells, which may be beneficial to homing of these cells to the BM environment.     

Th17 cells in systemic lupus erythematosus share functional features with Th17 cells from normal bone marrow and peripheral tissues

This study was designed to investigate the functional heterogeneity of human Th17 and how their plasticity shapes the nature of immune cell responses to inflammation and autoimmune diseases, such as systemic lupus erythematosus (SLE). We evaluated functional Th17 cell subsets based on the profile of cytokine production in peripheral blood (PB), bone marrow aspirates (BM) and lymph node biopsies (LN) from healthy individuals (n = 35) and PB from SLE patients (n = 34). Data were analysed by an automated method for merging and calculation of flow cytometric data, allowing us to identify eight Th17 subpopulations. Normal BM presented lower frequencies of Th17 (p = 0.006 and p = 0.05) and lower amount of IL-17 per cell (p = 0.03 and p = 0.02), compared to normal PB and LN biopsies. In the latter tissues were found increased proportions of Th17 producing TNF-α or TNF-α/IL-2 or IFN-γ/TNF-α/IL-2, while in BM, Th17 producing other cytokines than IL-17 was clearly decreased. In SLE patients, the frequency of Th17 was higher than in control, but the levels of IL-17 per cell were significantly reduced (p < 0.05). Among the eight generated subpopulations, despite the great functional heterogeneity of Th17 in SLE, a significant low proportion of Th17 producing TNF-α was found in inactive SLE, while active SLE showed a high proportion producing only IL-17. Our findings support the idea that the functional heterogeneity of Th17 cells could depend on the cytokine microenvironment, which is distinct in normal BM as well as in active SLE, probably due to a Th1/Th2 imbalance previously reported by our group.     

Variations in the four and a half LIM domains 1 gene (<Emphasis Type="Italic">FHL1</Emphasis>) are associated with fasting insulin and insulin sensitivity responses to regular exercise

Aims/hypothesis The expression of the four and a half LIM domains 1 gene (FHL1) is increased in the muscle of individuals who show an improvement in insulin sensitivity index (S _I) after 20 weeks of exercise training. The aim of the present study was to investigate associations between three FHL1 single nucleotide polymorphisms (SNPs) and variables derived from an IVGTT, both in the sedentary state and in response to exercise training, in participants in the HERITAGE Family Study. Materials and methods SNPs were typed using fluorescence polarisation methodology. Analyses were performed separately by sex and in black and white individuals. Results In black participants, no associations were found with any of the SNPs. In white women (n = 207), SNP rs9018 was associated with the disposition index (D _I), which is calculated as S _I generated from the MINMOD program (×10⁻⁴ min⁻¹[μU/ml]⁻¹) multiplied by acute insulin response to glucose (AIR_g; pmol/l × 10 min), and the glucose disappearance index (K _g) training responses (p = 0.016 and p = 0.008, respectively). In white men (n = 222), all SNPs were associated with fasting glucose levels (p ≤ 0.05) and SNP rs2180062 with the insulin sensitivity index (S _I) (p = 0.04) in the sedentary state. Two SNPs were associated with fasting insulin training response. Fasting insulin decreased to a greater extent in carriers of the rs2180062 C allele (p = 0.01) and rs9018 T allele (p = 0.04). With exercise training, S _I (×10⁻⁴ min⁻¹[μU/ml]⁻¹: 0.68 ± 0.20 vs −0.77 ± 0.44, p = 0.046), D _I (319 ± 123 vs –528 ± 260, p = 0.006) and K _g (per 100 min: 0.09 ± 0.04 vs −0.14 ± 0.8, p = 0.03) improved more in the C allele carriers at rs2180062 than in the T allele carriers. Conclusions/interpretation Fasting insulin and S _I responses to exercise training were associated with DNA sequence variation in FHL1 in white men. Whether these associations exist only in white men remains to be investigated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the <Emphasis Type="BoldItalic">FLT3</Emphasis> internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin⁻, HLA-DR⁺, CD11c⁺, CD86⁺) and plasmacytoid DC (pDC: Lin⁻, HLA-DR⁺, CD123⁺, CD86⁺) in diagnostic samples of 47 FLT3-ITD⁻ and 40 FLT3-ITD⁺ AML patients. The majority of ITD⁺ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD⁻ AML samples. Interestingly, mDCs and pDCs sorted out from ITD⁺ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD⁺ AML (n = 11) and ITD⁻ AML (n = 12) samples analyzed for mixed lineage DCs (Lin⁻, HLA-DR⁺, CD11c⁺, CD123⁺). ITD⁺ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.     

Correlation between granulocyte/macrophage-colony-forming units and CD34+ cells in apheresis products from patients treated with different chemotherapy regimens and granulocyte-colony-stimulating factor to mobilize peripheral blood progenitor cells

We examined the efficiency of disease-specific “standard” chemotherapies epirubicin, cyclophosphamide (EC); cyclophosphamide, vincristine, doxorubicin, etoposide, prednisolone (CHOEP); epirubicin, ifosfamide (EPI/IFOS) for peripheral blood progenitor cell (PBPC) mobilization in comparison to well-characterized mobilization protocols, i.e. etoposide, ifosfamide, cisplatin, epirubicin (VIPE) and dexamethasone, carmustine, etoposide, cytarabine, melphalan (DexaBEAM). Twenty-seven patients with various malignancies underwent 75 apheresis procedures for PBPC collection. Median cell yields from all 75 aphereses were 1.18 × 10⁵ mononuclear cells/kg [range (0.28–3.7) × 10⁸], 1.4 × 10⁵ granulocyte/macrophage-colony-forming units (CFU-GM)/kg [range (0.2–11) × 10⁵] and 3.3 × 10⁶ CD34⁺cells/kg [range (0.35–17.7) × 10⁶. CD34⁺/CD90⁺ cells could be mobilized by all mobilization regimens used. The difference observed in the mobilization of CD34⁺ cells was only of low significance when the mobilization regimens were compared, whereas the mobilizations of MNC and CFU-GM were significantly different between the groups. Breast cancer patients treated with the VIPE regimen (including pretreated women) had a significantly higher CFU-GM rate than patients treated with EC (P = 0.0005). Mobilized CD34⁺ PBPC were correlated with CFU-GM in all apheresis products. The linear correlation coefficients differed for the various mobilization groups: DexaBEAM (r=0.9, P < 0.0001), VIPE (r = 0.68, P = 0.0024), CHOEP (r = 0.52, P = 0.022), EPI/IFOS (r=0.34, P=0.11) and EC (r=0.23, P=0.2). We conclude that clonogenic assays can provide additional information about the autotransplant quality, particularly when alternative or new mobilization regimens are being investigated. Received: 7 January 1998 / Accepted: 24 February 1998     

Anti-agalactosyl IgG antibodies in Thai patients with rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis

This study was performed to determine the prevalence of anti-agalactosyl IgG antibodies in Thai patients with RA, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and determine the sensitivity and specificity of anti-agalactosyl IgG antibodies in the diagnosis of RA in comparison with IgM-rheumatoid factor (IgM-RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies. Serum samples were obtained from 100 patients with RA, 50 cases of SLE, 50 cases of SSc, and 100 healthy controls and analyzed for the presence of anti-agalactosyl IgG antibodies, IgM-RF and anti-CCP antibodies. A serum value greater than mean + 2 standard deviation of normal value of anti-agalactosyl IgG antibodies and anti-CCP antibodies was considered positive. The prevalence of anti-agalactosyl IgG antibodies in RA, SLE, and SSc patients was 88.0%, 14.0%, and 12.0%, respectively. The serum level of anti-agalactosyl IgG antibodies in patients with RA (227.10 ± 353.64 AU/mL) was significantly higher than those in SLE (11.84 ± 52.04 AU/mL), SSc (18.85 ± 99.60 AU/mL), and healthy controls (2.14 ± 1.97 AU/mL), (p < 0.001). There was a good correlation between the log serum level of anti-agalactosyl IgG antibodies and IgM-RF (r = 0.92, p < 0.001), anti-CCP antibodies and IgM-RF (r = 0.49, p < 0.001), and anti-agalactosyl IgG antibodies and anti-CCP antibodies (r = 0.55, p < 0.001). The sensitivity and specificity in the diagnosis of RA was 88.00% and 96.00% for anti-agalactosyl IgG antibodies, 90.00% and 99.00% for anti-CCP antibodies, and 91.00% and 95.00% for IgM-RF, respectively. The serum level of anti-agalactosyl IgG antibodies was significantly higher in RA than in SLE, SSc, and healthy controls. There was a good correlation between serum levels of anti-agalactosyl IgG antibodies, anti-CCP antibodies, and IgM-RF. These three tests had comparable sensitivity and specificity.     

Tandem high-dose therapy in relapsed and refractory B-cell lymphoma: results of a prospective phase II trial of myeloablative chemotherapy,followed by escalated radioimmunotherapy with <Superscript>131</Superscript>I-anti-CD20 antibody and stem cell rescue

A phase II trial evaluated safety, feasibility and efficacy of a sequential tandem approach combining myeloablative BEAM chemotherapy and autologous stem cell transplantation (ASCT) with myeloablative radioimmunotherapy (HD-RIT), with ¹³¹I-anti-CD20 antibody (¹³¹I-rituximab), followed by a second ASCT in patients with relapsed or refractory CD20+ B-cell lymphoma. According to protocol, 16 patients with relapsed (n = 14) and refractory (n = 2) CD20+ B-cell lymphoma received salvage therapy with rituximab and Dexa-BEAM, followed by BEAM (HD chemotherapy) and high-dose myeloablative radioimmunotherapy 2–6 months after BEAM. Nine of 16 patients received HD-RIT; seven patients were excluded before HD-RIT because of toxicity or progressive disease. Disease histologies were follicular lymphoma (FL) grades 1 and 2 (n = 4), transformed follicular (FL 3b; n = 6), diffuse large B-cell (DLBCL; n = 4), mantle cell (n = 1) and marginal zone lymphoma (n = 1). After a median follow-up of 50.4 months for OS and 39.7 months for progression-free survival (PFS), estimated 4-year OS and PFS were 67% and 64%, respectively. The estimated 4-year OS and PFS for patients with FL were 80% and 78%, respectively. Toxicity was significant, including one fatal outcome due to pneumonitis. Tandem transplants consisting of HD chemotherapy followed by HD-RIT with ¹³¹I-coupled anti-CD20 are manageable and effective but toxic treatment modalities for relapsed poor prognosis CD20+ B-NHL.     

Successful mobilization of peripheral blood stem cells in heavily pretreated myeloma patients with G-CSF alone

 We investigated the feasibility of mobilizing peripheral blood stem cells (PBSC) with G-CSF alone in 24 patients with multiple myeloma. The median age was 53 years (range 33–62). All patients had stage II/III disease and responded to standard first-line (n=6) or salvage chemotherapy (n=18). The median number of previous chemotherapy cycles was 7 (4–18) and the median number of prior melphalan-cycles was 6 (0–14). Nine (35%) patients had experienced prior radiation therapy. The patients received either 10 μg/kg G-CSF (n=18) or 24 μg/kg G-CSF (n=7, including one patient with previous 10 μg/kg G-CSF stimulation) daily s.c. for 5 or more consecutive days until completion of harvesting, starting apheresis on the fifth day. G-CSF treatment was well tolerated, with only slight bone pain in half of the patients (51%). After a median of three (range 1–7) apheresis procedures, medians of 3.8 (0.3–17)×10⁶ CD34+ cells/kg, 8.5 (4.5–24)×10⁸ MNC/kg, 2.9 (0.6–39.4)×10⁴ CFU-GM/kg, and 5.6 (0.9–49)×10⁴ BFU-E/kg were harvested. Three patients (12%) with extensive melphalan pretreatment failed the target collection of at least 2.0×10⁶ CD34+ cell/kg. Pretreatment with six or more cycles of melphalan yielded a smaller number of CD34+ cells than pretreatment with fewer than six cycles (2.5 vs 5.3×10⁶/kg;p=0.001). Nineteen patients underwent high-dose chemotherapy consisting of either total marrow irradiation (9 Gy)/busulfan (12 mg/kg) and cyclophosphamide (120 mg/kg) (n=10), or busulfan (14 mg/kg)/cyclophosphamide (120 mg/kg) (n=5), or tandem melphalan (200 mg/m²). The median time for granulocyte (>1.0/nl) and platelet (>50/nl) recovery was 10 and 14 days (ranges 7–12 and 8–40), respectively. G-CSF alone is a safe, alternative approach to mobilizing sufficient PBSC in patients with multiple myeloma and allows an exact prediction of harvest time. G-CSF-mobilized PBSCs ensure rapid engraftment after myeloablative therapy. Melphalan treatment should be avoided in patients who are candidates for high-dose chemotherapy. Received: February 5, 1998 / Accepted: April 14, 1998     

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

In the present study, we questioned whether the cholesterol synthesis inhibitor lovastatin potentiates the cytotoxicity of chemotherapeutic agents in the primitive CD34⁺ subpopulation of acute myeloid leukemia (AML) cells. AML mononuclear cells (n = 17) were sorted in CD34⁺ and CD34⁻ fractions and compared to normal CD34^+/− cells (n = 7). The percentage of surviving cells upon exposure to lovastatin (25–100 μM) and/or chemotherapeutics (cytarabin or daunorubicin) was determined with a luminescent cell viability assay. The results demonstrate that the primitive CD34⁺ subpopulation of normal and AML cells displayed a higher sensitivity to lovastatin than the more mature CD34⁻ subpopulation. The combination of lovastatin and chemotherapeutics resulted in a more pronounced inhibitory effect on both subpopulations. In contrast to the homogeneous results in normal CD34⁺ cells, a distinct heterogeneity in lovastatin sensitivity was found in AML samples. Therefore, a group of normal (n = 11) and abnormal (n = 6) responders were identified based on a reduced or increased cell survival compared to normal CD34⁺ cells. This distinction was not only observed in the CD34⁺ AML subfraction but also in CD34⁺CD38⁻AML cells. In the abnormal responder group, 50% of patients presented with unfavorable cytogenetics and significant higher peripheral blast cell counts, which coincided with poor treatment results. In summary, the findings indicate that the primitive subfraction of CD34⁺ AML cells is in the majority of cases affected by lovastatin treatment, which is potentiated when combined with chemotherapeutics. Heterogeneity of the response observed in AML patients allowed identification of a subgroup with poor prognosis.     

IgG deposits can be detected in cell nuclei of patients with both lupus erythematosus and malignancy

The purpose was to find immunological disturbances in lupus erythematosus (LE) patients with concomitant malignancy. 159 LE patients have been analyzed. Routine laboratory analyses including screening of serum autoantibodies and analyzing peripheral blood mononuclear cells by using flow cytometry have been performed. Malignant diseases have been revealed in 12 (7.5%) cases. All patients suffered from internal malignancies. LE patients with vs without malignancy had significantly decreased anti-double stranded DNA (16.6 vs 31.6%; p < 0.05) and increased anti-SSA/SSB (83.3 vs 32.2%/26.4%; p < 3 × 10⁻¹²) antibodies. Patients with neoplastic disease had increased IgG within the cell nuclei (76.6% ± 9.6 vs 51.8 ± 4.6%; p < 2 × 10⁻⁷). IgG penetrating living cells has been shown previously in SLE but has so far not been found in association to LE patients with malignant disease.     

Influence of Mast Cells on the Expression of Adhesion Molecules on Circulating and Migrating Leukocytes in Acute Pancreatitis-Associated Lung Injury

Pancreatitis-associated lung injury is an early-occurring and severe complication, still associated with substantial mortality. A number of inflammatory cells and their products are involved in the initiation and progress of the condition. In the present study, acute pancreatitis (AP) was induced by the intraductal infusion of 5% sodium taurodeoxycholate in the rat. Pulmonary endothelial barrier dysfunction was measured by plasma exudation of radiolabeled albumin. Expression of PECAM-1, ICAM-1, and L-selectin on neutrophils (CD11b⁺) and monocytes/macrophages (CD11b/c⁺), obtained from circulation and lung tissue, was measured 1 and 6 hours after AP induction (n = 10 rats/time point/group). Plasma levels of histamine and serotonin were determined. The role of mast cells was evaluated by pretreatment with the mast cell stabilizer cromolyn. Intraperitoneal administration of cromolyn downregulated pancreatitis-induced systemic increase of histamine at 1 hour (513 ± 82 vs. 309 ± 50, p < 0.05). Cromolyn prevented a decreased expression of PECAM-1 on circulatory neutrophils and monocytes/macrophages and against an increased expression of ICAM-1 and PECAM-1 on pulmonary neutrophils and monocytes/macrophages 6 hours after AP induction (about 40% vs. 10%, p < 0.01). The mast cell stabilizer also prevented pancreatitis-induced pulmonary endothelial barrier dysfunction at 6 hours. Thus, our data indicate that mast cells may play a critical role in the activation of leukocytes during the initiation of pancreatitis-associated lung injury by altering phenotypes of adhesion molecules.     

NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

Human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment. These changes are more frequently found in CD8+ T cells, and there are not well-defined markers of differentiation in the CD4+ subset. Typical features of cell immunosenescence are characteristics of pathologies in which the aberrant expression of NKG2D in CD4+ T cells has been described. To evaluate a possible age-related expression of NKG2D in CD4+ T cells, we compared their percentage in peripheral blood from 100 elderly and 50 young adults. The median percentage of CD4+ NKG2D+ in elders was 5.3% (interquartile range (IR): 8.74%) versus 1.4% (IR: 1.7%) in young subjects (p < 0.3 × 10⁻¹⁰). CD28 expression distinguished two subsets of CD4+ NKG2D+ cells with distinct functional properties and differentiation status. CD28+ cells showed an immature phenotype associated with high frequencies of CD45RA and CD31. However, most of the NKG2D+ cells belonged to the CD28^null compartment and shared their phenotypical properties. NKG2D+ cells represented a more advanced stage of maturation and exhibited greater response to CMV (5.3 ± 3.1% versus 3.4 ± 2%, p = 0.037), higher production of IFN-γ (40.56 ± 13.7% versus 24 ± 8.8%, p = 0.015), lower activation threshold and reduced TREC content. Moreover, the frequency of the CD4+ NKG2D+ subset was clearly related to the status of the T cells. Higher frequencies of the NKG2D+ subset were accompanied with a gradual decrease of NAIVE and central memory cells, but also with a higher level of more differentiated subsets of CD4+ T cells. In conclusion, CD4+ NKG2D+ represent a subset of highly differentiated T cells which characterizes the senescence of the immune system.     

Early Increase in Myocardial Perfusion After Stem Cell Therapy in Patients Undergoing Incomplete Coronary Artery Bypass Surgery

Incomplete revascularization is associated with worse long-term outcomes. Autologous bone marrow cells (BMC) have recently been tested in patients with severe coronary artery disease. We tested the hypothesis that intramyocardial injection of autologous BMC increases myocardial perfusion in patients undergoing incomplete coronary artery bypass grafting (CABG). Twenty-one patients (19 men), 59 ± 7 years old, with limiting angina and multivessel coronary artery disease (CAD), not amenable to complete CABG were enrolled. BMC were obtained prior to surgery, and the lymphomonocytic fraction separated by density gradient centrifugation. During surgery, 5 mL containing 2.1 ± 1.3 × 10⁸ BMC (CD34+ = 0.8 ± 0.3%) were injected in the ischemic non-revascularized myocardium. Myocardial perfusion was assessed by magnetic resonance imaging (MRI) at baseline and 1 month after surgery. The increase in myocardial perfusion was compared between patients with <50% (group A, n = 11) with that of patients with >50% (group B, n = 10) of target vessels (stenosis ≥ 70%) successfully bypassed. Injected myocardial segments included the inferior (n = 12), anterior (n = 7), and lateral (n = 2) walls. The number of treated vessels (2.3 ± 0.8) was significantly smaller than the number of target vessels (4.2 ± 1.0; P < 0.0001). One month after surgery, cardiac MRI showed a similar reduction (%) in the ischemic score of patients in group A (72.5 ± 3.2), compared to patients in group B (78.1 ± 3.2; P = .80). Intramyocardial injection of autologous BMC may help increase myocardial perfusion in patients undergoing incomplete CABG, even in those with fewer target vessels successfully treated. This strategy may be an adjunctive therapy for patients suffering from a more advanced (diffuse) CAD not amenable for complete direct revascularization.     

Association Between CD4<Superscript>+</Superscript>, Viral Load,and Pulmonary Function in HIV

Purpose

The antiretroviral therapy era has shifted the epidemiology of HIV-associated diseases, increasing the recognition of non-infectious pulmonary complications secondary to HIV. We aimed to determine the association between CD4⁺, viral load, and pulmonary function in individuals with uncontrolled HIV, and determine how changes in these parameters are associated with pulmonary function longitudinally.

Methods

This is a retrospective observational study of individuals with HIV who underwent pulmonary function testing in an urban medical center between August 1997 and November 2015.

Results

Of the 146 participants (mean age 52 ± 10 years), 49% were Hispanic, 56% were men, and 44% were current smokers. CD4⁺ <200 cells/μl was associated with significant diffusion impairment compared to CD4⁺ ≥200 cells/μl (DL_CO 56 vs. 70%, p = <0.01). VL (viral load) ≥75 copies/ml was associated with significant diffusion impairment compared to VL <75 copies/ml (DL_CO 60 vs. 71%, p = <0.01). No difference in FEV1, FEV1/FVC, or TLC was noted between groups. In univariate analysis, CD4⁺ and VL correlated with DL_CO (r = +0.33; p = <0.01; r = ?0.26; p = <0.01) and no correlation was noted with FEV₁, FEV₁/FVC, or TLC. Current smoking and history of AIDS correlated with DL_CO (r = ?0.20; p = 0.03; r = ?0.20; p = 0.04). After adjusting for smoking and other confounders, VL ≥75 copies/ml correlated with a 11.2 (CI 95% [3.03–19.4], p = <0.01) decrease in DL_CO. In Spearman’s Rank correlation, there was a negative correlation between change in VL and change in DL_CO over time (ρ = ?0.47; p = <0.01).

Conclusion

The presence of viremia in individuals with HIV is independently associated with impaired DL_CO. Suppression of VL may allow for recovery in diffusing capacity over time, though the degree to which this occurs requires further investigation.

     

Repeated Low-dose of Erythropoietin is Associated with Improved Left Ventricular Function in Rat Acute Myocardial Infarction Model

Objective To evaluate the potential protective affects of Epo on left ventricular (LV) function and remodeling after acute myocardial infarction (MI). Methods Epo was injected into the peritoneum of male Wistar rats (250 g) during 6 weeks post induction of MI. Rats were divided into five groups: MI treated with single high dose (MT1, 5,000 U/kg, n = 10), single high dose (5,000 U/kg) and repeated high doses (MTHi, 1,000 U/kg twice a week; n = 8), or single high dose (5,000 U/kg) and repeated low doses (MTLo, 750 U/kg once a week, n = 10), MI non-treated (MNT, n = 10), sham (S, n = 5). Echocardiography was performed 3.6 ± 1.5 days and 43.7 ± 2.3 days post MI. Collagen deposition and infarct size were measured on histological sections using computerized image analysis. Apoptosis was assessed by ApopTag staining. Results Baseline fractional shortening (FS) was similar between groups. Six weeks after MI the FS of MTLo (26.9%) was significantly higher compared to MNT (17.8%), MT1 (19.5%) and MTH (22.3%) (p = 0.01). However, remodeling indices (end diastolic and end systolic areas, LV circumference) did not improve in the Epo groups, and even worsened in the MTHi group. There was significantly less collagen staining in non-infarct areas in MT1 and MTHi groups compared to MNT and MTLo (0.38 ± 0.3%, 0.49 ± 0.34%, vs 0.89 ± 0.41%, 0.95 ± 0.33%, respectively, p < 0.001). The number of ApopTag positive nucleus was significantly higher in the MNT group compared to the MT1, MTHi, MTLo groups (14.4 ± 8, 7.6 ± 4, 5.8 ± 7, 4.8 ± 5, respectively, p = 0.01 for trend). Conclusion Repeated low doses of Epo after MI improved LV function, but the role of Epo on remodeling is not clear. It did not reduce left ventricular indices, but reduces fibrosis and apoptosis. High Epo doses reduced LV function and aggravated remodeling.