Minimal residual disease negativity in elderly patients with acute myeloid leukemia may indicate different postremission strategies than in younger patients.

In the present analysis, we evaluated whether in elderly acute myeloid leukemia (AML) patients (>60 years), minimal residual disease (MRD) assessed by flow cytometry may have a role in guiding choice of postremission strategies. We analyzed 149 young and 61 elderly adults who achieved morphological CR after induction course of EORTC/GIMEMA protocols. Elderly patients reached a postconsolidation MRD negative status less frequently than younger ones (11 vs 28 %, p = 0.009). MRD negativity resulted in a longer 5-year disease-free survival (DFS) both in elderly (57 vs 13 %, p = 0.0197) and in younger patients (56 vs 31 %, p = 0.0017). Accordingly, 5-year cumulative incidence of relapse (CIR) of both elderly (83 vs 42 %, p = 0.045) and younger patients (59 vs 24 % p = NS) who were MRD positive doubled that of MRD negative ones. Nevertheless, CIR of MRD negative elderly patients was twofold higher than that of younger MRD negative ones (42 vs 24 %, p = NS). In conclusion, elderly patients in whom chemotherapy yields a MRD negative CR have duration of DFS and rate of CIR significantly better than those who remain MRD positive. Nonetheless, the high CIR rate observed in the elderly suggests that MRD negativity might have different therapeutic implications in this population than in the younger counterpart.

Production and characterization of monoclonal antibodies against major histocompatibility complex class I chain-related gene A.

Major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand for the activating immunoreceptor natural killer group 2D (NKG2D), is expressed on stressed cells such as tumor cells. Study of expression of this molecule on tumor cells and patients' sera is useful to define patients' stages leading to proper selection of therapy. In this study, mouse anti-MICA monoclonal antibodies (mAbs) were produced by DNA immunization using a gene gun. Screening of anti-MICA-producing mouse and hybridomas were performed by immunoblot and cell enzyme-linked immunosorbent assay (ELISA) against MICA-positive HeLa and -negative Me1386 cell lines. MAbs were characterized against MICA-positive and -negative cell lines by immunoblot, cell ELISA and flow cytometry. The mAbs were also characterized for locus and allele specificities of MICA and MHC class I chain-related gene B (MICB) as well as for their ability to stain formalin-fixed paraffin-embedded tissues by immunohistochemistry. Although all mouse immune sera were positive with MICA-positive cells by both immunoblot and cell ELISA methods, some hybridomas were positive only with one method. The mAbs had diverse specificities to detect MICA and MICB and different abilities to stain formalin-fixed paraffin-embedded tissues. Thus, DNA immunization by gene gun is an effective method to generate immune mice for the production of mAbs with a variety of specificities against native and denatured forms of MIC proteins.

Phenotype and function of a CD56+ peripheral blood monocyte.

G-CSF primed CD34 cells cultured for 2-3 weeks in IL-2 and stem cell factor generate CD56(high) cells with phenotypic and morphologic features of NK cells, and a novel adherent CD56(low) CD16- population expressing myeloid markers (CD33 and HLA-DR). We hypothesized that similar cells might also occur in peripheral blood. In 13/13 normal individuals, we found a circulating population of CD56(low), CD33+, FcgammaRI+, FcgammaRII+, HLA-DR+, CD11b(high), CD14+ monocytes closely resembling the cultured CD56(low)CD33+ cells. They may represent a normal counterpart of the CD56+ CD33+ hybrid myeloid/natural killer cell leukemia. Their mean frequency was 1.3+/-1% (standard deviation), range 0.16-3.5%, of total mononuclear cells. CD56(low)CD33+ cells, primed with cytomegalovirus antigen, induced autologous T-lymphocyte proliferation comparably to CD56-, CD14+ peripheral blood monocytes (PBM). Conversely, CD56(low) cells induced greater T-cell proliferation than CD56- PBM when lymphocyte responders were HLA mismatched. Unstimulated CD56(low)CD33+ cells showed a low antiproliferative effect on K562, which was increased upon LPS stimulation. The pattern of cytokine production by CD56(low)CD33+ cells and PBM largely overlapped; however, they produced detectable levels of IL-6 and IL-1beta. These results define a minor monocyte population with distinct phenotypic and functional features.

SGK-1 protects kidney cells against apoptosis induced by ceramide and TNF-α.

Ceramide regulates several different cellular responses including mechanisms leading to apoptosis. Serum- and glucocorticoid-inducible protein kinase (SGK)-1 is a serine threonine kinase, which activates survival pathways in response to stress stimuli. Recently, we demonstrated an anti-apoptotic role of SGK-1 in human umbilical endothelial cells treated with high glucose. In the present study, since ceramide induces apoptosis by multiple mechanisms in diabetes and its complication such as nephropathy, we aimed to investigate whether SGK-1 may protect even against apoptosis induced by ceramide in kidney cells. Human embryonic kidney (HEK)-293 cells stable transfected with SGK-1 wild type (SGK-1wt) and its dominant negative gene (SGK-1dn) have been used in this study. Apoptotic stimuli were induced by C2-ceramide and TNF-α to increase endogenous synthesis of ceramide. Upon activation with these stimuli, SGK-1wt transfected cells have a statistically significant reduction of apoptosis compared with SGK-1dn cells (P<0.001). This protection was dependent on activation of caspase-3 and Poly-ADP-ribose-polymerase-1 (PARP-1) cleavage. SGK-1 and AKT-1 two highly homologous kinases differently reacted to ceramide treatment, since SGK-1 increases in response to apoptotic stimulus while AKT-1 decreases. This enhancement of SGK-1 was dependent on p38-mitogen-activated-protein kinases (p38MAPK), cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide-3-kinase (PI3K) pathways. Especially, by using selective LY294002 inhibitor, we demonstrated that the most involved pathway in the SGK-1 mediated process of protection was PI3K. Treatment with inhibitor of SGK-1 (GSK650394) significantly enhanced TNF-α-dependent apoptosis in HEK-293 cells overexpressing SGK-1wt. Caspase-3, -8 and -9 selective inhibitors confirmed that SGK-1 reduced the activation of caspase-dependent apoptosis, probably by both intrinsic and extrinsic pathways. In conclusion, we demonstrated that in kidney cells, overexpression of SGK-1 is protective against ceramide-induced apoptosis and the role of SGK-1 can be potentially explored as a therapeutic target in conditions like diabetes, where ceramide levels are increased.

Tissue-restricted T cell alloresponses across HLA barriers: selection and identification of leukemia-restricted CTL in HLA-mismatched stimulator-responder pairs.

Exploiting the graft-versus-leukemia (GVL) effect in mismatched transplants requires its separation from graft-versus-host disease (GVHD). We generated leukemia-specific cytotoxic T lymphocytes (CTL) in three haplotype-mismatched, two class I-mismatched and two single HLA-A locus-matched stimulator-responder pairs. Six patients with chronic myelogenous leukemia and one patient with acute myeloid leukemia transformed from MDS were studied. CTL generated after 10 days stimulation with unselected leukemic peripheral blood mononuclear cells inhibited leukemic CFU-GM colony growth (>85% at 10:1 effector:target ratio) with no third-party colony inhibition. In five pairs, responders were cultured separately with leukemia cells, PHA-B or LCL from the stimulator. After 2-4 restimulations, the T cell repertoire was examined by flow analysis using Vbeta-specific antibodies. Test cultures (but not controls) showed preferential expansion of 1-4 Vbeta families either common to two or more stimulators or unique to a particular stimulator. Notably, we elicited leukemia-specific TCR Vbeta expansions on four out of five occasions. In two pairs, responder cells selected for the appropriate leukemia-specific Vbeta family were shown to have leukemia-specific cytotoxicity. These leukemia-restricted T-cells were CD8+ or CD4+ and CD25+ or CD57+. The results support the development of strategies to selectively deplete GVHD and conserve GVL reactivity in mismatched transplants.

[Synergism between cyclosporin and gangliosides in anti-rejection therapy. Experimental in vivo model]. / Sinergismo tra ciclosporina e gangliosidi nella terapia antirigetto. Modello sperimentale in vivo.

In vivo gangliosides (GS) bring about inhibitory effects on the immune response which is attributable to a weakening of the interaction between interleukin-2 (IL-2) and its receptor. In a previous paper we showed that the GS are capable of bringing about in vitro the immunosuppressive power of cyclosporin A CyA2 in this paper we have tried to associate gangliosides (GS) and low dose of CyA in the grafting of rat allogen cutis in order to have in this way in vivo a confirmation of the results previously obtained in vitro. Cutaneous strips taken from Lewis rats were grafted into Sprague Dawley rats and treated for 21 days with intraperitoneal administration of GS, of Cya, and a mixture of the two. The rats were not treated and the rats treated with GS and with CyA separately rejected the graft of the cutis. On the other hand, the use of an association of Gs and CyA brought about a successful graft in 8 rats out of 10. Splenic cells extracted 21 days after the graft from rats treated with GS and with CyA separately were stimulated in vitro with Packweed mitogen (PWM); on the contrary the cells extracted from rats treated with a combination of the two drugs did not react to the stimulation with PWM. Our results show that the GS brings about in vivo the immunosuppressive effects of low doses of CyA.

Cytokine response to ischemia/reperfusion injury in an ex vivo perfused porcine liver model.

BACKGROUND: We evaluated the degree of inflammatory response after ischemia/reperfusion injury by an extracorporeal normothermic autologous hemoperfusion of porcine livers. MATERIALS AND METHODS: Livers explanted from 7 pigs were perfused extracorporeally at 39 degrees C with autologous blood. Serum samples were obtained hourly until 6 hours from the beginning of reperfusion and assayed for 9 different cytokines. RESULTS: Significant elevations in interleukin 6 (IL-6) and IL-8 were noted following reperfusion (P < .001), with both demonstrating an increase which followed a sigmoid curve; other cytokines that were assessed showed no significant change. CONCLUSIONS: The ex vivo model excludes the liver from the influence of external systemic factors such as hormones, the autonomic nervous system, and other regulatory molecules produced elsewhere in the body, allowing the response to the ischemia/reperfusion injury to be studied in isolation and in considerable detail. Although this study examined a relatively short period, the increases in only IL-6 and IL-8 suggested that these are important molecules in the early phase after reperfusion.

"Systemic apoptotic response" after thermal burns.

The systemic pathophysiologic changes following thermal injuries affect multiple organs and body systems leading to clinical manifestations including shock, intestinal alterations, respiratory and renal failure, immunosuppression and others. Recent advances in the comprehension of mechanisms underlying systemic complications of thermal injuries have contributed to uncover part of the cellular and molecular basis that underlie such changes. Recently, programmed cell death (apoptosis) has been considered playing an important role in the development of such pathological events. Therefore, investigators utilizing animal models and clinical studies involving human primates have produced a large body of information suggesting that apoptosis is associated with most of the tissue damages triggered by severe thermal injuries. In order to draw the attention on the important role of apoptosis on systemic complications of thermal injuries, in this review we describe most of these studies, discuss possible cellular and molecular mechanisms and indicate ways to utilize them for the development of therapeutic strategies by which apoptosis may be prevented or counteracted.

Transfer of PR1-specific T-cell clones from donor to recipient by stem cell transplantation and association with GvL activity.

BACKGROUND: The curative effects of GvL following transfer of donor-derived T cells during allogeneic stem cell transplantation (SCT) are well established. However, little is known about the nature, origin and kinetics of the anti-leukemic T-cell responses involved. METHODS: We used quantitative real-time PCR (qRT-PCR) for interferongamma mRNA production (IFN-gamma) and PR1/HLA-A*0201 tetramer staining to detect PR1-specific CD8+ T-cell activity in a donor and a patient with CML. Unbiased strand switch anchored RT-PCR was used to further characterize specific clones in PR1 sorted CD8+ T-cell populations. RESULTS: We identified PR1-specific CD8(+) T-cell clones from a donor pre-transplant, and demonstrated their transfer in the recipient's blood post-SCT using molecular tracking of Ag-specific T-cell receptors. PR1-specific CD8(+) T-cell populations were polyclonal, with a range of functional avidities for cognate Ag, and displayed predominantly effector memory phenotype early post-SCT, suggesting active stimulation in vivo. Expansion of these PR1-specific CD8(+) T-cell clones in the recipient was followed by complete remission of CML. DISCUSSION: This report represents the first direct demonstration that PR1-specific CD8(+) T-cell clones can be transferred during SCT, and supports the feasibility of pre-transplant vaccination strategies that aim to boost the number of anti-leukemic T cells in the graft.

CD44 is a cytotoxic triggering molecule in human peripheral blood NK cells.

Previous studies have shown that target cells that bind to CD44 adhesion molecules on cloned cytotoxic T cells are lysed by the CTL. To determine whether CD44 is also a cytotoxic trigger molecule in human PBL, we tested a bispecific Ab consisting of anti-CD44 Fab cross-linked to a Fab against a target cell Ag, in cytotoxicity assays using PBL as effectors. We found that PBL mediated lysis in the presence of the anti-CD44 bispecific Ab provided that the effector cells were stimulated with either IL-2 or IL-12. Cell fractionation experiments showed that CD44-directed cytolysis was mediated exclusively by CD56+ low buoyant density cells, mainly by NK (CD16+) cells, but also to a lesser extent by CD56+ T cells. CD44-directed cytolysis appeared in these subsets 24 to 48 h after addition of IL-2 and paralleled the acquisition of Ab-independent (LAK) activity; in contrast, these cells mediated Ab-dependent cellular cytotoxicity and CD3 redirected lysis before stimulation with IL-2. Unstimulated CD56+ cells uniformly expressed high levels of CD44 that increased modestly after incubation with IL-2. No changes in isoform expression in the extracellular domain of CD44 could be detected upon activation with the use of isoform-specific mAbs. Thus, lymphokine stimulation caused CD44 to become a cytotoxic trigger in subsets of PBL that mediated other forms of cytotoxicity and expressed CD44 before activation, suggesting that activation of these cells was accompanied by a coupling of CD44 to their lytic machinery.

Signaling pathways regulating CD44-dependent cytolysis in natural killer cells.

CD44 is a cytotoxic triggering molecule on activated, but not fresh natural killer (NK) cells. In the current study, metabolic pathways used in CD44-directed lysis (CD44DL) were examined using activated human NK cells as effectors. We found that CD44 expressed by activated NK cells was indistinguishable in isoform and molecular weight from CD44 on unactivated cells. However, de novo protein expression was required for the induction of CD44DL, suggesting that activated NK cells contain proteins not present in fresh NK cells that couple CD44 to the lytic machinery. Concanimycin A, a selective inhibitor of perforin-based cytolysis, totally blocked CD44DL, natural cytotoxicity, and antibody-dependent cell-mediated cytolysis (ADCC). Moreover, studies in which kinase inhibitors were added during the effector phase of lysis indicated that protein-tyrosine and ser/thr kinases were required for all three cytolytic activities and that protein kinase C played a nonessential role in lysis. By contrast, wortmannin totally inhibited CD44DL, but failed to block natural cytotoxicity and only partially blocked ADCC, suggesting that phosphatidylinositol 3-kinase (PI 3-kinase) is required at an early, receptor-specific stage of CD44DL. Finally, cytochalasin B enhanced CD44DL, but not ADCC, indicating that CD44DL is modulated by actin polymerization. Taken together, our data suggest that CD44 in NK cells interacts with proteins induced during interleukin-2 activation in a triggering pathway that induces perforin release, requires PI 3-kinase, and is modulated by the cytoskeleton.

Alternative triggering molecules and single chain bispecific antibodies.

Although T cell receptors and Fc receptors are the best known cytotoxic triggering molecules, a number of adhesion molecules recently have been identified as alternative triggering molecules. We discuss how CD44, an adhesion molecule found on all leukocytes and many other cell types, becomes a triggering molecule on NK cells following stimulation with IL-2. We also describe a genetically engineered single chain bispecific antibody, produced in mammalian cells and in bacteria, that is capable of redirecting lysis in the nanogram per milliliter range.

Immunocompromised tumor-bearing mice show a selective loss of STAT5a/b expression in T and B lymphocytes.

Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.