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PubMed2000

Antibodies against advanced glycation end product Nepsilon-(carboxymethyl)lysine in healthy controls and diabetic patients.

AIMS/HYPOTHESIS: Nepsilon-(carboxymethyl)lysine (CML) is one of the end products of protein glycoxidation and its accumulation is associated with diabetes complications. Since CML-modified proteins are immunogenic, we have investigated the presence of anti-CML antibodies in diabetic patients. METHODS: Antibodies against CML-modified human serum albumin (HSA) were measured by direct enzyme-linked immunosorbent assay in the sera from 289 non-selected diabetic and in 120 healthy control subjects. RESULTS: Immunoglobulin-G reactivity towards CML-HSA was significantly higher in diabetic than in control sera. The presence of anti-CML IgG in diabetics, however, was not influenced by age, duration of disease or glycaemic control. Analysis of distribution frequency revealed that anti-CML IgG in both control and diabetic subjects were not normally distributed and that the distribution curves were similar in the two groups. Moreover, only 14% of the diabetic subjects displayed antibody binding to CML-HSA above 95 centile in the control cohort. Competition experiments confirmed that the IgG detected in both control and diabetic groups were specific for CML epitopes and did not recognise glycated-HSA in which CML formation was inhibited. CONCLUSION/INTERPRETATION: The presence of anti-CML IgG in diabetic sera is probably not related to the development of an immune response against protein glycoxidation products.

Adult↗

PubMed2005

Myeloperoxidase cytochemical negativity: an unexpected but intrinsic property of blasts of all phases of chronic myeloid leukemia.

Myeloperoxidase (MPO) cytochemical activity, recognized as a very important hallmark of myeloblasts, is generally negative in chronic myeloid leukemia (CML) blast crisis (BC). Because this finding is unexpected, being not in keeping with the myeloproliferative nature of CML, we tried to ascertain if MPO cytochemical negativity could be an intrinsic property of blasts of CML and hence present in the preblastic phases as well. Myeloperoxidase cytochemistry of peripheral blood blasts in 161 cases of CML, including 103 in chronic phase (CP) and 29 each in accelerated phase (AP) and BC, was assessed and compared with that of 30 cases of acute myeloid leukemia, AML-M2. Blasts of 97 (94.2%) of 103 cases of CP, 28 (96.6%) of 29 cases of AP, and 22 (75.9%) of 29 cases of BC were negative for MPO (<3% MPO-positive blasts). Compared with the strong MPO positivity, both in terms of intensity and proportion, in the AML-M2 cases, the positivity in the CML cases was generally weak and was seen in a small number of blasts (5-15%), except in one case of BC with 20% positive blasts. Absence or, at times, weak MPO cytochemical activity is an intrinsic property of blasts of all phases of CML, and use of the term myeloblast in CML should be understood to refer to a cell with this property. This also explains why MPO cytochemistry, despite its high reputation as a myeloid-lineage marker, generally does not help in CML BC. CML BC should therefore be considered as a possible diagnosis along with acute lymphoblastic leukemia, AML-M0, AML-M7, etc., in the setting of MPO-negative blasts. Similarity between MPO expression pattern in CML, i.e., negative in blasts and positive in the more mature cells, and that during maturation of normal myeloid series of cells shows the deranged myelopoiesis of CML to be undisturbed at least with respect to MPO expression. There is need for a more comprehensive study of blasts of preblastic phases.

Biomarkers, Tumor↗

PubMed2003

Identification of Nepsilon-(carboxymethyl)lysine-positive cells in astroglia-rich primary cultures.

Astroglia-rich primary cultures from rat brain were used to investigate the presence in glial cells of Nepsilon-(carboxymethyl)lysine (CML), an advanced glycation endproduct. Westernblot analysis of homogenates of rat brain as well as of astroglia-rich cultures demonstrated the presence of CML-modified proteins in these samples. Immunocytochemical staining of astroglia-rich cultures revealed that only a minority of the cells in these cultures were intensively stained for CML. The staining intensity of CML-positive cells was strongly reduced, if the cells were not permeabilized, indicating that intracellular proteins were CML-modified. The CML-positive cells were identified as astrocytes and oligodendrocytes by double-labelling immunocytochemical staining for CML and the cellular markers galactocerebroside, myelin basic protein and glial fibrillary acidic protein. In contrast to other glial cells, microglial cells in astroglia-rich cultures were CML-negative. The finding that only a minority of cells in astroglia-rich cultures contains high amounts of intracellular CML-modified proteins indicates that specific properties of these CML-positive cells are responsible for the CML-formation in these cells.

Animals↗

PubMed1994

Leukemia cell lines: in vitro models for the study of chronic myeloid leukemia.

The clinical importance of CML lies in its poor responsiveness to chemotherapy which has proved highly effective in treating ALL. The scientific importance of CML resides in its role as a cancer prototype, permitting the identification of genes centrally involved in both neoplastic change and normal cellular differentiation. One of these genes, the fusion gene BCR/ABL resulting from the balanced translocation (9;22) has received wide attention owing to its intimate involvement in CML. Although a tremendous amount of data have been recently discovered about BCR/ABL, its exact role in leukemogenesis and normal hematopoiesis remains obscure. The study of CML cell lines has already been of considerable help in understanding the molecular events associated with the Ph chromosome [4]. Further advances are likely to be forthcoming, particularly at the molecular genetic, but also at the protein level. CML cell lines may offer an excellent means of addressing many issues as continuous cell lines represent an inexhaustible source of identical cell material that, in addition, can be made available to other researchers around the world. This overview on the thus far reported CML-derived cell lines supports the hypothesis that in some specimens of CML the target cells in which Ph translocation arises are not necessarily lineage-restricted committed progenitor cells, but are in fact in some (or all?) cases precommitted bipotential or multipotential progenitor or stem cells retaining the potential for differentiation in diverse hematopoietic directions [26]. In conclusion, established tumor cell lines with their unique phenotypic and karyotypic features have been extremely useful models for investigation of the molecular and biological characteristics of CML. Considerable progress in understanding the molecular and cell biology of CML has been achieved. Further advances in the knowledge of CML are expected to accrue with the productive use of these powerful research tools for many important unresolved issues. By so doing, these discoveries might open new avenues that promise to move clinicians closer to the goal of the prevention or cure of CML in all patients.

Adult↗

PubMed1998

Immunohistochemical localization of different epitopes of advanced glycation end products in human atherosclerotic lesions.

To better understand the role of advanced glycation end products (AGEs) in atherogenesis, we developed specific antibodies against different immunological epitopes of AGE structures, including Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML), and demonstrated the immunohistochemical localization of CML- and nonCML-epitopes in atherosclerotic lesions of human aorta, which were obtained at autopsy from 20 nondiabetic patients (12 males and eight females; mean age, 60.8+/-16.7 years). Monoclonal anti-CML antibody (6D12) recognized not only AGE-modified proteins, but also CML-modified proteins. On the other hand, polyclonal anti-nonCML antibody reacted to AGE-modified proteins, but not to CML-modified proteins. Both antibodies were unreactive to the early-stage products of glycation, including fructose-modified butyloxycarbonyl-lysine and fructose-epsilon-aminocaproic acid. Atherosclerotic lesions included diffuse intimal thickening (DIT), fatty streaks (FS), atherosclerotic plaques (AP) and complicated lesions. An immunohistochemical analysis showed both CML- and nonCML-epitopes to be found along the collagen fibers in DIT in subjects more than 40 years old, but not in subjects less than 40 years old. CML-epitopes accumulated mainly in the cytoplasm of macrophage/foam cells, while nonCML-epitopes accumulated exclusively in the extracellular spaces in FS. APs showed the CML-epitope stored macrophage/foam cells, and the accumulation of both CML- and nonCML-epitopes in the lipid-rich fibrous area. An immunohistochemical analysis with a monoclonal antibody against oxidized low density lipoprotein (FOH1a/DLH3) showed the presence of this antigen within the cytoplasm of the macrophage/foam cells in atherosclerotic lesions, which were also positive for the CML-epitopes. These findings thus suggest that the heterogeneous localization of AGEs in atherosclerotic lesions depends on their different epitopes, and that a close link, therefore, exists between the peroxidation of LDL and the formation of AGEs in atherosclerotic lesions.

Adult↗

PubMed2003

Flow cytometric analysis of aneuploidy and S-phase fraction in chronic myeloid leukemia patients: role in early detection of accelerated phase.

We studied S-phase fraction (SPF) and aneuploidy in peripheral blood leucocytes of patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP, n=41), accelerated phase (CML-AP, n=6), and control subjects (n=12) with an aim to find out their role in early detection of accelerated phase. The SPF and aneuploidy were studied through flow-cytometry using LT-Mod. Fit software. Mean SPF value in CML-AP (9.28+/-3.46%) and in CML-CP (4.76+/-2.30%) were significantly higher than in normal controls (0.28+/-0.21%), (P<0.005, P<0.001). CML-CP patients having higher SPF (>7%) converted to accelerated phase within 18 months of follow-up while those with lower SPF (<7%) did not. Aneuploidy was present in 34.14% of CML-CP and all patients of CML-AP whereas no control subjects showed aneuploidy. Among CML-CP patients having SPF >7%, 86% developed aneuploidy during follow-up as compared to 18.50% of CML-CP with less than 7% SPF. We conclude that peripheral blood SPF and aneuploidy could be important parameters for prediction of evolution to accelerated phase in CML patients.

Adult↗

PubMed2000

CD14+ peripheral blood mononuclear cells from chronic myeloid leukemia and normal donors are inhibitory to short- and long-term cultured colony-forming cells.

Monocyte-induced cell-cytotoxicity has been implicated in the mechanism of suppression of normal haematopoietic progenitors in chronic myeloid leukemia (CML). We examined here the in vitro effect of CML-derived and normal peripheral blood (PB) monocytes on short- and long-term cultured haematopoietic progenitor cells. Short-term coculture (5 days) of CML or normal monocytes with CML or normal peripheral blood mononuclear cells (PBMNC)/CD34+ cells as targets resulted in a significant inhibition of colony-forming cell (CFC) growth. Coculture conditioned medium (CCM) from 5-days cocultures of normal or CML CD14+ monocytes with CD34+ cells were likewise inhibitory to CFC. In 5-week long-term cocultures of monocytes in direct contact with normal bone marrow (BM) progenitors, CML monocytes reduced the proportion of long-term cultured CFC (LTC-CFC) significantly to 52% of the controls, while normal monocytes had a less pronounced inhibitory effect (89% of the controls) on LTC-CFC. Reduction of LTC-CFC was great when CML monocytes and target cells were separated by a transwell membrane as compared to control cultures in the absence of CD14+ cells (53.5 vs. 9%). CCM from 5-week cocultures of normal or CML CD14+ monocytes with CD34+ progenitors from bone marrow (BM) cells were also inhibitory to CFC. No difference in cytokine levels for TNF-alpha, IFN-gamma, G-CSF, IL-10, IL-6 was detectable between CML CD14+ CCM and control CCM derived from short- and long-term cocultures. Our results suggest that CML monocytes may play a role in the inhibition of normal haematopoiesis through a yet not defined soluble factor supporting the expansion of the malignant clone in CML.

Adult↗

PubMed2000

Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation.

We investigated whether primary chronic myelogenous leukemia (CML) hematopoietic progenitors demonstrated altered proliferation and maturation in response to growth factor (GF) stimulation. The effect of GF stimulation on proliferation and expansion of committed and primitive progenitors (colony forming cells [CFC]) was evaluated. Culture of CML and normal CD34(+) cells with different GF for 7 days resulted in similar expansion of committed progenitors (CFC). In contrast, GF culture conditions that expanded normal primitive progenitors (week-6 long-term culture-initiating cells (LTC-IC)] led to depletion of CML LTC-IC numbers. GF culture also resulted in increased depletion of week-10 extended LTC-IC, which represent an even more primitive progenitor population, from CML compared with normal CD34(+) cells. CML CD34(+) cells enter into cycle more quickly than normal CD34(+) cells and CML CFC expansion was accelerated compared to normal CFC. Evaluation of primitive progenitor proliferation using PKH-26 and single-cell LTC-IC analysis demonstrated that the majority of CML LTC-IC remaining after GF culture originated from divided CD34(+) cells, whereas GF-cultured normal LTC-IC were derived mainly from undivided cells. Depletion of CML primitive progenitor numbers in association with increased proliferation suggests increased sensitivity to GF-induced maturation. These studies indicate that CML primitive progenitors have enhanced sensitivity to GF-induced cell division and maturation. Altered GF responsiveness may contribute to abnormal expansion of malignant myeloid cells in CML. These findings may also be applied toward the development of novel approaches to select benign stem cells in CML.

Antigens, CD34↗

PubMed2003

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects.

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).

Adult↗

PubMed1998

Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors.

Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.

Cell Adhesion↗

PubMed2004

Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

Androstadienes↗

PubMed2000

Retroviral coexpression of IFN-alpha and IFN-gamma genes and inhibitory effects in chronic myeloid leukemia cells.

Interferon (IFN) is an effective treatment for chronic myeloid leukemia (CML) in chronic phases, and a number of in vitro antileukemic effects of IFN on CML cells have been reported. The transfer of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. We first investigated the possibility of transducing CML cells with the retroviral vectors LIalpha2SN and LIgammaSN, encoding the IFN-alpha2 and IFN-gamma genes, respectively, and with the bicistronic vector LIalpha2IrIgammaSN coexpressing the IFN-alpha2 and IFN-gamma genes. We then analyzed the effects of IFN-alpha2 and IFN-gamma produced alone or simultaneously on the proliferation of CML cells. We optimized the transduction efficiency by using the CML-derived K562 cell line. We then introduced IFN genes into CML CD34+ cells. Secretion of IFN-alpha and IFN-gamma was demonstrated in K562 and CML CD34+ cells transduced with the different vectors. The MHC class I antigens were overexpressed in both K562 and CML CD34+ transduced cells. Inhibition of the proliferation of LIalpha2IrIgammaSN-transduced CML cells was greater than with the LIalpha2SN and the LIgammaSN-transduced CML cells. We demonstrate an additive effect of IFN-alpha and IFN-gamma on the inhibition of K562 and CML CD34+ cell proliferation.

Antigens, CD34↗

PubMed2004

Nepsilon-(carboxymethyl)lysine, Nepsilon-(carboxyethyl)lysine and vascular cell adhesion molecule-1 (VCAM-1) in relation to peritoneal glucose prescription and residual renal function; a study in peritoneal dialysis patients.

BACKGROUND: Advanced glycation end products (AGEs) may contribute to peritoneal and cardiovascular damage in peritoneal dialysis (PD) patients, possibly in part by over-expression of vascular cell adhesion molecule-1 (VCAM-1). It has been suggested that peritoneal glucose load, oxidative stress, as well as the uraemic state itself may lead to an increased formation of AGEs. Aims of the present study were first to investigate the relation between residual glomerular filtration rate (rGFR), malondialdehyde (MDA) as a marker of lipid peroxidation, and peritoneal glucose prescription and absorption with serum levels of VCAM-1 and with the well characterized AGEs N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL), as well as with CML and CEL in peritoneal effluent. METHODS: CML and CEL were measured by tandem mass spectroscopy, MDA by HPLC, and VCAM-1 by ELISA in 37 stable PD patients (age 54 +/- 12 years; time on PD 25 +/- 18 months). CML and CEL were also measured after a 4-month interval. RESULTS: rGFR was independently related to CML both in serum (r = -0.66; P<0.001) and effluent (r = -0.62; P<0.001), whereas peritoneal glucose prescription and absorption were, respectively, related to CML in serum and effluent (r = 0.49; P<0.001 and r = 0.44; P<0.01). Relationships were comparable when assessed after the follow-up period. Peritoneal glucose absorption (r = 0.37; P<0.05), but not rGFR, was related to CEL in serum. The relation between peritoneal glucose prescription and CML in effluent lost significance when rGFR was added to the multi-regression model. Both rGFR (r = -0.40; P<0.05) and peritoneal glucose absorption (r = 0.37; P<0.05) were associated with VCAM-1 expression, which was itself weakly related only to CML in effluent (r = 0.38; P<0.05). MDA was not related to any parameter. CONCLUSION: Peritoneal glucose prescription and absorption, as well as rGFR are related to serum and effluent levels of CML and to VCAM-1 expression in serum, whereas peritoneal glucose absorption was related to serum levels of CEL. Still, the effect of rGFR, which does not appear to be mediated through lipid peroxidation pathways, on effluent levels of CML appears to outweigh the effect of the PD treatment. Even small differences in residual renal function in patients already on dialysis therapy are related to large variations of CML in serum and the peritoneal cavity.

Cross-Sectional Studies↗

PubMed1984

Suppression of lymphocyte reactivity by blood transfusions in uremic patients. III. Regulation of cell-mediated lympholysis.

It has previously been demonstrated that blood transfusions (BT) can induce the generation of suppressor cells in the mixed lymphocyte reaction (MLR). We have further investigated whether regulation of the alloimmune response could also be observed at the cell-mediated lympholysis (CML) level. We have, therefore, prospectively analyzed the effect of the first 2 BT on cytotoxic T lymphocyte (CTL) differentiation in previously nontransfused uremic patients. Different patterns of CML changes were noted. In 7 cases, marked CML reduction was observed after the first BT, and in 7 others CML remained unchanged or even increased after the first BT, but reduced CML occurred after the second. Increased CML was noted in the last 4 cases. When present, reduction in CML after BT could indeed occur upon in vitro restimulation by cells of the specific donor, but also by those of other individuals regardless whether they shared HLA antigens. Post-BT lymphocytes the CML of which was decreased could inhibit CML of autologous pre-BT cells when used as an irradiated third-party at the initiation of the sensitization step. No correlation, however, was generally observed between MLR and CML suppressions when both were tested in parallel assays. CML reduction could occur in the presence of unchanged or increased MLR, and no cytotoxicity to the stimulating cells could be observed in direct cytolysis assays, so this inhibition can be attributed to suppressor cells of CTL differentiation that are generated in vivo after BT in man.

Blood Transfusion↗

PubMed1992

The time course of cell-mediated lympholysis in rat hepatic allograft recipients pretreated with a single donor-specific blood transfusion.

A single intravenous injection of 1 ml freshly heparinized donor blood seven days before transplantation significantly prolonged the survival of subsequent donor-specific hepatic allografts in the fully allogeneic ACI(RT1a-to-LEW(RT1l)) rat combination. The time course of cell-mediated lympholysis was studied in this animal model. The activity of CML in lymphocytes infiltrating into the hepatic allograft (CML-G) and in the spleen (CML-S) was determined by measuring % lysis of donor Con A blast cervical lymph node cells. Preoperative DST resulted in an increased activity of CML-S with a peak (31.6%, E/T = 150) on day 7. This increased CML-S activity after DST rapidly declined during the first days following hepatic transplantation. The activities of both CML-S and CML-G then increased after transplantation and reached peaks on days 15 (48%, E/T = 150) and 20 (2.57%, E/T = 75), respectively. These were much higher than the peak values of CML-S (11.2%, E/T = 150) on day 7 and CML-G (19.5%, E/T = 75) on day 6 in untreated controls and were followed by a subsequent gradual decrease in those activities to preoperative levels by day 113 posttransplant. Phenotypic analysis of lymphocytes infiltrating grafts in DST-treated hosts demonstrated that the CD4/CD8 ratio remained relatively constant (less than 1.0). While the ratio in control grafts increased and reached a peak (2.17) on day 9. Histological examination revealed that mononuclear cell infiltration of grafts reached a peak on day 9 in both DST-treated hosts and controls. This mononuclear cell accumulation gradually subsided in DST-enhanced grafts. The mitotic index of graft hepatocytes reached a peak on day 15 in DST-treated hosts and on day 7 in control. The evidence of prolonged survival of hepatic grafts in recipients pretreated with DST, despite the presence of cytotoxic T cells with increased CML activity in vitro, suggests that effector cytotoxic cell activity may not be necessary for rat liver allograft rejection and that there may be limitations in measuring host cytotoxic activity simply by CML assays.

Animals↗

PubMed2001

BCR rearrangement-negative chronic myelogenous leukemia revisited.

PURPOSE: To document the characteristics of patients with major breakpoint cluster region (M-bcr) rearrangement-negative chronic myelogenous leukemia (CML). PATIENTS AND METHODS: The hematopathologist, who was blinded to patients' molecular status, reviewed the referral bone marrows and peripheral-blood smears from 26 patients with Philadelphia (Ph) translocation-negative CML who lacked Bcr rearrangement (and other evidence of a Bcr-Abl anomaly) and 14 patients (controls) with chronic-phase Ph-positive CML. Clinical data was ascertained by chart review. RESULTS: Among the 26 M-bcr rearrangement-negative CML patients, three pathologic subtypes emerged: (1) patients indistinguishable from classic CML (n = 9), (2) patients with atypical CML (n = 8), and (3) patients with chronic neutrophilic leukemia (n = 9). Among the 14 patients with Ph-positive CML who were included in the blinded review, 13 were classified as classic CML, and one was classified as atypical CML. The only statistically significant difference between M-bcr rearrangement-negative subgroups was in the proportion of patients having karyotypic abnormalities, an observation common only in patients with atypical CML (P = 0.008). However, the small number of patients in each subgroup limited our ability to differentiate between them. Interferon alfa induced complete hematologic remission in five of 14 patients; four of these remissions lasted more than 5 years. Only one of 26 patients developed blast crisis. The median survival of the 26 patients was 37 months. CONCLUSION: Patients with M-bcr rearrangement-negative CML fall into three morphologic subgroups. Disease evolution does not generally involve blastic transformation. Instead, patients show progressive organomegaly, leukocytosis, anemia, and thrombocytosis. Some patients in each subgroup can respond to interferon alfa.

Adult↗

PubMed1993

Role of aberrant sialylation of chronic myeloid leukemia granulocytes on binding and signal transduction by chemotactic peptides and colony stimulating factors.

Chronic myelogenous leukemia (CML) granulocytes exhibit a number of characteristics attributable to immature granulocytes, including marked increases in cell surface sialylation of glycoproteins which may be due, at least in part, to an increased activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:Ga1 beta 1-3Ga1NAc alpha(2-3)-sialyltransferase (EC 2.4.99.4), and perhaps to altered activity of other glycosyltransferases and sialidases. This aberrant sialylation of CML granulocytes contributes to the decreased binding of the synthetic chemotactic peptide, formyl Met Leu Phe (fMLP), to the surface of CML granulocytes which leads to a rapid, transient increase in cytosolic free calcium ([Ca2+]i), an integral step in the biochemical cascade leading to cell activation. To determine if the decrease in binding of fMLP to CML granulocytes translates into a functional deficit, we measured fMLP-induced increases in [Ca2+]i. Compared to normal granulocytes, fMLP-induced increases in [Ca2+]i were markedly decreased in CML granulocytes. After sialidase treatment, a significant augmentation in fMLP-induced increases in [Ca2+]i was noted in CML granulocytes, indicating that the decreased signalling may be a consequence of aberrant sialylation. To determine if the effects of aberrant sialylation also alters the binding of endogenous polypeptide mediators, we determined the effect of desialylation of CML and normal granulocytes on binding of the colony stimulating factor for granulocytes and monocytes (GM-CSF), which plays a role in differentiation and proliferation of myeloid-lineage cells. As with fMLP binding, we also showed that the binding of GM-CSF to CML granulocytes, but not normal granulocytes, was markedly increased after sialidase treatment. Similarly, binding of GM-CSF to undifferentiated HL-60 cells was markedly increased after sialidase treatment. Therefore, we have demonstrated that aberrant sialylation of CML granulocytes not only alters the binding of fMLP and GM-CSF to their receptor(s), but may also alter signal transduction. Thus, aberrant glycosylation of CML granulocytes may reduce the binding of hematopoietic growth factors, which in turn may be responsible for the immature phenotype of CML granulocytes.

Calcium↗

PubMed2000

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation.

Cell Division↗