Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus.

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.

Rapid turnover of T lymphocytes in SIV-infected rhesus macaques.

Studies of lymphocyte turnover in animal models have implications for understanding the mechanism of cell killing and the extent of lymphocyte regeneration in human immunodeficiency virus infection. Quantitative analyses of the sequential changes in bromodeoxyuridine labeling of CD4 and CD8 T lymphocytes not only revealed the normal proliferation and death rates of these cell populations in uninfected macaques, but also showed a substantial increase in these rates associated with simian immunodeficiency virus (SIV) infection. Faster labeling and delabeling in memory and naïve T lymphocyte subpopulations as well as in NK (natural killer) and B cells were also observed in infected macaques, suggesting a state of generalized activation induced by SIV.

Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.

High-dose chemotherapy using BEAM without autologous rescue followed by reduced-intensity conditioning allogeneic stem-cell transplantation for refractory or relapsing lymphomas: a comparison of delayed versus immediate transplantation.

Patients with refractory/relapsing lymphoma are rarely cured by chemotherapy. High-dose chemotherapy (HDC) for tumor debulking followed by reduced-intensity conditioning (RIC) hematopoietic stem-cell transplantation (HSCT) has been advocated as a concept. We previously treated 10 patients (group A) with BEAM chemotherapy followed by delayed RIC HSCT at day 28. We now report on the subsequent 11 patients receiving BEAM followed immediately by fludarabine/total body irradiation and allogeneic HSCT (group B), and compare the outcome to group A patients. Non-hematological toxicity before engraftment was comparable, only gut toxicity was higher in group B. Days in aplasia, days on antibiotics and length of hospital stay were significantly longer in group A. Cumulative incidence of acute (GvHD) >or=grade II and incidence of chronic GvHD were lower in group B. At last follow-up, seven patients in group A were alive, with six of them in complete remission. In group B, nine patients were alive, seven of them in complete remission. No significant difference in estimated 3-year overall survival was seen. These data challenge the initial concept of debulking first and delaying allogeneic RIC HSCT. Allogeneic HSCT with standard BEAM conditioning is a valid alternative for patients with resistant/relapsed lymphoma, which might be considered earlier in the disease course.

Combination of broad molecular screening and cytogenetic analysis for genetic risk assignment and diagnosis in patients with acute leukemia.

We evaluated the impact of genetic analysis combining cytogenetics and broad molecular screening on leukemia diagnosis according to World Health Organization (WHO) and on genetic risk assignment. A two-step nested multiplex RT-PCR assay was used that allowed the detection of 29 fusion transcripts. A total of 186 patients (104 males (56%), 174 adults (94%), 12 children (6%), 155 AML (83%), 31 ALL (17%)) characterized by morphology and immunophenotyping were included. Of these 186 patients, 120 (65%) had a genetic abnormality. Molecular typing revealed a fusion transcript in 49 (26%) patients and cytogenetic analysis revealed an abnormal karyotype in 119 (64%). A total of 27 (14%) cases were genetically classified as favorable, 107 (58%) intermediate and 52 (28%) unfavorable. For 38 (20%) patients, there was a discrepancy in the genetic risk assignments obtained from broad molecular screening and cytogenetics. Cryptic fusion transcripts in nine (5%) patients changed the genetic risk assignment in four and the WHO classification in four patients. In 34 patients (18%), cytogenetics defined the risk assignment by revealing structural and numerical chromosomal abnormalities not detected by molecular screening. Broad molecular screening and cytogenetics are complementary in the diagnosis and genetic risk assignment of acute leukemia.

Activated natural killer cells from patients with acute myeloid leukemia are cytotoxic against autologous leukemic blasts in NOD/SCID mice.

Natural killer (NK) cells are implicated in the surveillance of hematological malignancies. They participate in the immune response against residual acute myeloid leukemia (AML) after hematopoietic stem cell transplantation with partial HLA class I disparity. However, the role of NK cells in autologous leukemia-specific immunity remains poorly understood. We studied the function of NK cells in AML patients at diagnosis. Following isolation, CD56+CD3- cells exhibited a high proliferative potential in vitro in response to interleukin (IL)-2. The polyclonal population of activated AML-NK cells expressed normal levels of the activating receptor NKG2D and the major natural cytotoxicity receptor NKp46. AML-NK cells were highly effective with respect to interferon-gamma production, cytotoxicity against HLA class I-deficient K562 erythroleukemia cells in vitro and retardation of tumor growth in vivo in K562-bearing NOD/SCID mice. Importantly, when AML blasts were injected into NOD/SCID mice, a single dose of adoptively transferred autologous AML-NK cells significantly reduced the AML load by 8-77%. Recognition of AML blasts may be related to the observed upregulation of ligands for NKG2D and natural cytotoxicity receptors in vivo. We conclude that AML patient-derived NK cells are fully functional, in support of exploring the benefit of AML immunotherapy with IL-2-stimulated autologous NK cells.

Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group.

Internal tandem duplications (ITDs) of the FLT3 gene have been observed in about 35% of APL cases. If FLT3-ITD is associated with a worse outcome in patients with acute myeloid leukemia (AML) in general, its prognostic value in acute promyelocytic leukemia (APL) is still a matter of debate. We investigated incidence, associated clinical features, and prognostic implication of FLT3-ITD, but also FLT3-D835 point mutation and N-Ras or K-Ras mutations in 119 APL patients, all prospectively enrolled in the two consecutive APL-93 and APL-2000 trials. Mutation incidences were 38, 20, and 4%, for FLT3-ITD, FLT3-D835, and Ras, respectively. The presence of FLT3-ITD was associated with high white blood cell count, high Sanz index, M3-variant subtype, and V/S PML-RAR alpha isoforms. Complete remission (CR), induction death, and death in CR rates were not affected by FLT3 or Ras mutations, as well as cumulative incidence of relapse. However, a trend for a shorter overall survival (P=0.09) was observed in FLT3-ITD patients, because of a very poor postrelapse survival (P=0.02). This feature, which has been also reported in patients with AML in general, is suggestive of an underlying genetic instability in FLT3-ITD patients, leading to the acquisition of additional unknown bad-prognosis gene mutations at relapse.

[Genetic diagnosis of acute leukemias--a practical guide]. / Genetische Diagnostik akuter Leukämien--ein praktischer Leitfaden.

Acute leukemias are the consequence of accumulation of immature precursor cells. Genetic alterations are cause of uncontrolled cell proliferation. They can be detected by conventional cytogenetics and molecular methods at diagnosis. Classification of acute leukemias according to the World Health Organization depend on the knowledge of the presence of these aberrations. Furthemore they are important prognostic parameters influencing therapy intensity. Genetic abnormalities can be targets of novel therapeutic strategies. Detection and monitoring of genetic abnormalities in acute leukemias are of major importance for correct management of patients.

The t(14;18) in a patient with de novo acute lymphoblastic leukemia is associated with t(8;9).

Cytogenetic analysis of a bone marrow aspirate from a patient with acute lymphoblastic leukemia (ALL) revealed the presence of a complex karyotype containing the translocation, t(14;18)(q32;q21). Further investigations using fluorescence in situ hybridization (FISH) allowed the characterization of an additional translocation, t(8;9)(q24;p1?). The association of t(14;18)(q32;q21) and t(8;9)(q24;p13) has recently been described in two patients with de novo ALL (Nacheva et al. Blood 1993;82:231-240) and this report supports these findings.

Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation.

This pilot study tested feasibility of natural killer cell purification and infusion (NK-DLI) in patients after haploidentical hematopoietic stem cell transplantation (HSCT). The aim was to obtain >or=1.0 x 10(7)/kg CD56+/CD3- NK cells and <1.0 x 10(5)/kg CD3+ T cells. Mononuclear cells were collected by 10 l leukapheresis. A two-step ex vivo procedure was used to purify NK cells, using an immunomagnetic T-cell depletion, followed by NK-cell enrichment. Five patients with high-risk myeloid malignancies were included, presenting 3-12 months after a haploidentical HSCT with mixed chimerism (3), impending graft failure (1) or early relapse (1). The purified product contained a median of 1.61 x 10(7)/kg (range 0.21-2.2) NK cells and 0.29 x 10(5)/kg (0.11-1.1) T cells. A purity of NK cells of 97% (78-99), a recovery of 35.5% (13-75), and a T-cell depletion of 3.55 log (2.9-4.5) was achieved. Infusions were well tolerated and none of the patients developed graft-versus-host disease. We observed an increase in donor chimerism in 2/5, stable mixed chimerism, decreasing chimerism and relapse of AML in one patient each. Selection of NK-DLI is technically feasible. NK cells are well tolerated when used as adoptive immunotherapy in recipients of haploidentical HSCT.

Expression patterns of the HIV type 1 coreceptors CCR5 and CXCR4 on CD4+ T cells and monocytes from cord and adult blood.

We have measured the surface expression of the HIV-1 coreceptors CCR5 and CXCR4 on CD4+ T cells and monocytes from cord and adult blood. The expression of CCR5 was largely restricted to the memory (CD45RAlow) subset, whereas CXCR4 was expressed on both memory and naive (CD45RAhigh) T cells. The paucity of memory CD4+ T cells in cord blood means that CCR5-positive cells are relatively uncommon, so the overall extent of CCR5 expression was reduced in cord blood, compared with adult blood. IL-2 activation of CD4+ T cells from both cord and adult bloods caused a substantial increase in CCR5 expression, but moderately decreased CXCR4 expression. PHA stimulation increased CCR5 expression slightly, but only on naive cells. Monocytes expressed both CCR5 and CXCR4 at levels that differed little between cord and adult blood.

High stem cell dose will not compensate for T cell depletion in allogeneic non-myeloablative stem cell transplantation.

The best strategies for non-myeloablative stem cell transplants (NST) are not known. We hypothesized that a high stem cell dose and post-transplant donor lymphocyte infusions (DLI) in a T cell-depleted NST setting may result in stable engraftment without severe GvHD. We used conditioning with 200 mg/kg cyclophosphamide, and ATG, a high peripheral stem cell dose of >10 x 10(6) CD34(+) cells/kg, T cell-depleted to <1 x 10(5) CD3(+) cells/kg followed by incremental DLI. Ten patients, 53 (42-61) years of age with hematological malignancy (CML in 3, MDS in 2, myeloma in 3 and CLL in 2) were included. All patients achieved initial engraftment, at a median 13.5 (10-20) days. Three patients achieved complete chimerism, four achieved a complete hematologic remission. In seven patients the graft ultimately failed. Acute GvHD grade II was seen in three patients after DLI. At a median follow-up of 28 months (range 15-35), eight patients are alive, none died of treatment-related complications. NST with T cell depletion to prevent GVHD results in a high graft failure rate. High stem cell dose (> or =10 x 10(6) CD34(+)cells/kg) and post-transplant DLI will not compensate for the lack of T cells to ensure stable engraftment.

Functional G-CSF pathways in t(8;21) leukemic cells allow for differentiation induction and degradation of AML1-ETO.

INTRODUCTION: Efficacy of differentiating agents requires that their specific cellular targets are still expressed and functional in the leukemic cells. One hypothesis to target sensitive cells is to select leukemic clones which harbor disrupted transcription factors. CBFalpha and CBFbeta are core-binding proteins which have been identified as transcription regulators of hematopoietic genes and shown to be altered in numerous leukemias. In M2 AML, the t(8;21) translocation, CBFalpha (AML1) is altered and produced as the AML1-ETO fusion protein. The fusion protein blocks transcription and differentiation mediated by G-CSF. Interestingly, AML1-ETO leukemic cell lines are sensitive to numerous cytokines in vitro and can be induced to differentiate in the presence of G-CSF and PMA. MATERIALS AND METHODS: As in the APL differentiation model, primary culture provides a useful tool for therapeutic screening of differentiation inducers, we analysed the in vitro sensitivity of 10 fresh M2 AML t(8;21) leukemic samples to G-CSF and the functionality of G-CSF intracellular pathways. In vitro data were compared with in vivo data from four patients treated with rhG-CSF at the dosage of 5 microg/kg/day i.v. for two to three weeks before the initiation of AML induction chemotherapy and immunophenotypic analysis performed weekly to monitor in vivo differentiation. RESULTS: In vitro, an increase in CD34+ cells expressing differentiation antigens (CD11b, CD13 or CD15) was noted along with a decrease of immature CD34+/differentiation antigen negative cells. After two weeks of a daily rhG-CSF administration in vivo, a significant, albeit transient, decrease of blast count was achieved, concomitant with an increase in differentiated leukemic cells suggesting that in vivo differentiation occurs. Fresh t(8;21) leukemic cells possess functional G-CSF signaling pathways as normal activity and kinetics of STAT1 and STAT3 binding was observed. Furthermore, differentiation induction leads to a subsequent degradation of the AML1-ETO oncoprotein. CONCLUSION: The data presented here supports the claim that G-CSF can induce in vitro and in vivo differentiation of M2 AML t(8;21) cells.

Spectrum of Reactivity with Three Monoclonal Antibodies-MHM6(CD23), L30(CD24) and UCHB1-in B-Cell Leukaemias.

Cells from patients with a range of B-cell leukaemias have been investigated for reactivity with three monoclonal antibodies-MHM6 (CD23), L30 (CD24) and UCHB1. Cells in suspension were studied by indirect immunofluorescence and fixed cells in cytospin preparations by an indirect immunoperoxidase technique. Positive results with CD23 were obtained in two thirds of patients with chronic lymphocytic leukaemia in contrast to one quarter of cases of other mature B-lineage leukaemias and lymphomas; cells of acute lymphoblastic leukaemia gave negative results. L30 (CD24) had a wide spectrum of reactivity within the B-lineage with negative reactions being common only in hairy cell leukaemia and multiple myeloma/plasma cell leukaemia. UCHB1 was most frequently and most strongly positive in prolymphocytic leukaemia. Our observations suggest that these reagents, in particular L30 (CD24) and MHM6 (CD23), provide useful additional information in the differential diagnosis of B-lineage lymphoproliferative disorders.

The calcium-binding protein calretinin-22k is detectable in the serum and specific cells of cancer patients.

BACKGROUND: Calretinin-22k (CR-22k), an alternatively spliced form of calretinin (CR) belongs to the EF-hand family of calcium-binding proteins and is expressed in several colon adenocarcinoma cell lines (e.g. WiDr, HT-29). MATERIALS AND METHODS: Serum samples of cancer patients were screened with a sandwich ELISA technique using the CR-specific antiserum 7696. Highly positive samples were analyzed by Western blots and immunohistochemistry. RESULTS: CR-22k was detected in the serum of several patients and values were as high as 0.19 microgram/ml. Western blot analysis confirmed the identity of the bound protein as CR-22k. The highest concentrations were detected in patients with colon or breast cancer, but also in a patient with ischemic necrosis of the gut. CR immunoreactivity was localized to epithelial cells, nerve fibres, cells of the connective tissue and to mesothelial cells. CONCLUSIONS: Our results establish that CR-22k is detectable in the serum of cancer patients under specific pathological conditions.

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers.

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40 and +100 after transplantation. Median doses (and ranges) of infused NK- and T-cells per product were 1.21 (0.3-3.8) × 10(7)/kg and 0.03 (0.004-0.72) × 10(5)/kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHDgrade II, all receiving a total of 0.5 × 10(5) T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.