C-Type Lectin-Like Molecule-1 as a Biomarker for Diagnosis and Prognosis in Acute Myeloid Leukemia: A Preliminary Study.

OBJECTIVE: AML is a heterogeneous disease both in genomic and proteomic backgrounds, and variable outcomes may appear in the same cytogenetic risk group. Therefore, it is still necessary to identify new antigens that contribute to diagnostic information and to refine the current risk stratification. METHODS: The expression of C-type lectin-like molecule-1 (CLL-1) in AML blasts was examined in 52 patients with newly diagnosed or relapsed/refractory AML and was compared with two other classic markers CD33 and CD34 in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML. Subsequently, the value of CLL-1 as a biomarker for prognosis was assessed in this malignant tumor. RESULTS: The results showed that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%) and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL-1 was able to complement the classic markers CD33 or CD34. After dividing the cases into CLL-1high and CLL-1low groups according to cutoff 59.0%, we discovered that event-free survival and overall survival (OS) of the CLL-1low group were significantly lower than that of the CLL-1high group, and low CLL-1 expression seems to be independently associated with shorter OS. CONCLUSIONS: These preliminary observations identified CLL-1 as a biomarker for diagnosis and prognosis of AML.

Endo180 modulation by bisphosphonates and diagnostic accuracy in metastatic breast cancer.

BACKGROUND: Endo180 (CD280; MRC2; uPARAP)-dependent collagen remodelling is dysregulated in primary tumours and bone metastasis. Here, we confirm the release and diagnostic accuracy of soluble Endo180 for diagnosing metastasis in breast cancer (BCa). METHODS: Endo180 was quantified in BCa cell conditioned medium and plasma from BCa patients stratified according to disease status and bisphosphonate treatment (n=88). All P-values are from two-sided tests. RESULTS: Endo180 is released by ectodomain shedding from the surface of MCF-7 and MDA-MB-231 BCa cell lines. Plasma Endo180 was significantly higher in recurrent/metastatic (1.71±0.87; n=59) vs early/localised (0.92±0.37; n=29) BCa (P<0.0001). True/false-positive rates for metastasis classification were: 85%/50% for the reference standard, CA 15-3 antigen (28 U ml(-1)); ≤97%/≥36% for Endo180; and ≤97%/≥32% for CA 15-3 antigen+Endo180. Bisphosphonate treatment was associated with reduced Endo180 levels in BCa patients with bone metastasis (P=0.011; n=42). True/false-positive rates in bisphosphonate-naive patients (n=57) were: 68%/45% for CA 15-3 antigen; ≤95%/≥20% for Endo180; and ≤92%/≥21% for CA 15-3 antigen+Endo180. CONCLUSION: Endo180 is a potential marker modulated by bisphosphonates in metastatic BCa.

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils.

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.

Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.

A study of effects of WGA and ConA on RBC membrane receptors using a new ektacytometric method.

With a new ektacytometry, we studied the relation between the microstructure of red blood cell (RBC) membrane and the rheological properties of RBCs in a shear flow field of low viscosity. The main contributions of this paper are as follows: 1. The hemorheological meanings of the orientation index (DI)or and the small deformation index (DI)d were explored. (DI)or is an overall rheological index depending on the deformability and morphology of RBCs. The better the physiological shape of RBCs is maintained, the greater the (DI)or is. (DI)d can be used to describe the lipid fluidity of RBC membrane. Such an explanation for the meaning of (DI)d has been forcefully supported by our experiments using electron spin resonance (ESR) and fluorescence polarization. 2. The influence of wheat germ agglutinin (WGA) of different concentrations on the lipid fluidity of membrane is different from that of concanavalin A (ConA). The lipid fluidity of membrane changes with WGA concentration treating RBCs and there is a maximum value for the membrane fluidity at a specific concentration of WGA. However, the deformability of membrane described by the integrate deformation index (IDI) monotonically decreased with the increase in WGA concentration treating RBCs. 3. It is concluded that the increase in the lipid fluidity of red cell membrane is not necessarily associated with the improvement of RBC deformability.

Interleukin-4 receptors on human blood mononuclear cells.

We have studied regulation of the expression of the interleukin-4 receptor (IL-4R) on human blood mononuclear cells (PBMC) using both 125I-IL-4 binding assay and flow cytometric analysis of biotinylated IL-4 (B-IL-4) binding. PBMC express approximately 300 high-affinity IL-4R per cell (Kd = 25-100 pM). Activation of PBMC for 60-80 hr by phytohemagglutinin (PHA) or concanavalin A (Con A) results in a 2- to 4.5-fold increase of IL-4R number without alteration of IL-4R affinity for IL-4. Binding of B-IL-4 showed that IL-4R expression is upregulated on virtually all PHA-stimulated PBMC, whereas it mostly concerns larger cells among Con A-activated PBMC. Reculture of PHA-blasts with 1 nM IL-4 further upregulates IL-4R expression to a level approximately 10-fold higher than observed on freshly isolated PBMC. Interestingly, IL-4 is able to reinduce high IL-4R levels on cells that have been deprived of IL-4 for 20 hr and IL-2 is almost as efficient. Finally, SDS-PAGE analysis of IL-4-binding molecules on unstimulated, PHA- and PHA/IL-4-activated PBMC revealed the same three peptides of MW 140-130, 80-75, and 70-65 kDa, as shown on human cell lines.

New abnormalities in the morphology, cell surface receptors, and electrolyte metabolism of In(Lu) erythrocytes.

The In(Lu) phenotype is inherited as an autosomal dominant trait and is characterized by suppression of the Lutheran, P1, i, and Aua erythrocyte blood group antigens. We have developed a monoclonal antibody (L21) that strongly agglutinates all erythrocytes except In(Lu), and we have identified eight In(Lu) individuals among 42,000 blood donors tested. Studies of two families confirmed the dominant mode of inheritance and revealed several new features of this phenotype. The erythrocytes of all five affected individuals from the two families exhibited diminished hemagglutination by the lectin concanavalin A, although they reacted normally with several other lectins. The erythrocytes of two affected individuals in one family exhibited marked acanthocytosis. The erythrocytes of the proposita of the other family exhibited a mild degree of poikilocytosis, but the cells of the other two affected individuals in this family had normal morphology. The osmotic fragility of fresh In(Lu) erythrocytes was normal, but after incubation for 24 hours at 37 degrees C in plasma the In(Lu) cells exhibited a marked increase in resistance to osmotic lysis. During the incubation period the erythrocytes lost K+ and their total cation content was diminished. These data indicate that in addition to the suppression of blood group antigens noted previously, the In(Lu) phenotype includes a variety of morphological abnormalities and a defect in electrolyte metabolism. The use of L21 and similar monoclonal antibodies provides a more sensitive means of detecting In(Lu) erythrocytes than typing with human anti-Lub antisera.

Plasma membrane carbohydrate composition and lectin receptors of lymphocytes from pro-lymphocytic leukaemia.

The carbohydrate composition of a purified plasma membrane fraction derived from the peripheral blood lymphocytes of a patient with a pro-lymphocytic leukaemia has been investigated and compared to the expression of lectin receptors at the membrane surface of the intact cell. The presence of galactose, mannose, fucose, glucose, sialic acid, N-acetylgalactosamine, N-acetylglucosamine and sialic acid substituted O-glycosidic linked beta-D-galactopyranosyl (1 leads to 3) N-acetyl-D-galactosamine was confirmed on membrane glycoconjugates by gas chromatography. A lectin induced agglutinin assay using intact leukaemia cells demonstrated the accessibility of different lectin receptors at the native membrane surface, while the peanut agglutinin and the Helix pomatia receptor was only accessible after neuraminidase treatment.