Plasmodium vivax inhibits erythroid cell growth through altered phosphorylation of the cytoskeletal protein ezrin.

BACKGROUND: The underlying causes of severe malarial anaemia are multifactorial. In previously reports, Plasmodium vivax was found to be able to directly inhibited erythroid cell proliferation and differentiation. The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and remain unclear. In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development. METHODS: This study describes the first comparative phosphoproteome analysis of growing erythroid cells (gECs), derived from human haematopoietic stem cells, exposed to lysates of infected erythrocytes (IE)/uninfected erythrocytes (UE) for 24, 48 and 72 h. This study utilized IMAC phosphoprotein isolation directly coupled with LC MS/MS analysis. RESULTS: Lysed IE significantly inhibited gEC growth at 48 and 72 h and cell division resulting in the accumulation of cells in G0 phase. The relative levels of forty four phosphoproteins were determined from gECs exposed to IE/UE for 24-72 h and compared with the media control using the label-free quantitation technique. Interestingly, the levels of three phosphoproteins: ezrin, alpha actinin-1, and Rho kinase were significantly (p < 0.05) altered. These proteins display interactions and are involved in the regulation of the cellular cytoskeleton. Particularly affected was ezrin (phosphorylated at Thr567), which is normally localized to gEC cell extension peripheral processes. Following exposure to IE, for 48-72 h, the ezrin signal intensity was weak or absent. This result suggests that phospho-ezrin is important for actin cytoskeleton regulation during erythroid cell growth and division. CONCLUSIONS: These findings suggest that parasite proteins are able to inhibit erythroid cell growth by down-regulation of ezrin phosphorylation, leading to ineffective erythropoiesis ultimately resulting in severe malarial anaemia. A better understanding of the mechanisms of ineffective erythropoiesis may be beneficial in the development of therapeutic strategies to prevent severe malarial anaemia.

Suppression of erythroid development in vitro by Plasmodium vivax.

BACKGROUND: Severe anaemia due to dyserythropoiesis has been documented in patients infected with Plasmodium vivax, however the mechanism responsible for anaemia in vivax malaria is poorly understood. In order to better understand the role of P. vivax infection in anaemia the inhibition of erythropoiesis using haematopoietic stem cells was investigated. METHODS: Haematopoietic stem cells/CD34+ cells, isolated from normal human cord blood were used to generate growing erythroid cells. Exposure of CD34+ cells and growing erythroid cells to P. vivax parasites either from intact or lysed infected erythrocytes (IE) was examined for the effect on inhibition of cell development compared with untreated controls. RESULTS: Both lysed and intact infected erythrocytes significantly inhibited erythroid growth. The reduction of erythroid growth did not differ significantly between exposure to intact and lysed IE and the mean growth relative to unexposed controls was 59.4 ± 5.2 for lysed IE and 57 ± 8.5% for intact IE. Interestingly, CD34+ cells/erythroid progenitor cells were susceptible to the inhibitory effect of P. vivax on cell expansion. Exposure to P. vivax also inhibited erythroid development, as determined by the reduced expression of glycophorin A (28.1%) and CD 71 (43.9%). Moreover, vivax parasites perturbed the division of erythroid cells, as measured by the Cytokinesis Block Proliferation Index, which was reduced to 1.35 ± 0.05 (P-value<0.01) from a value of 2.08 ± 0.07 in controls. Neither TNF-a nor IFN-g was detected in the culture medium of erythroid cells treated with P. vivax, indicating that impaired erythropoiesis was independent of these cytokines. CONCLUSIONS: This study shows for the first time that P. vivax parasites inhibit erythroid development leading to ineffective erythropoiesis and highlights the potential of P. vivax to cause severe anaemia.

Skin Anti-Aging Potential of Ipomoea pes-caprae Ethanolic Extracts on Promoting Cell Proliferation and Collagen Production in Human Fibroblasts (CCD-986sk Cells).

Collagen loss in the skin dermis is a major cause of age-related changes to the skin. Natural phytochemical substances are desirable for the prevention of skin aging and the formation of wrinkles. Ipomoea pes-caprae (IPC) has been utilized for nutritional and therapeutic purposes, and its extract contains collagenase inhibitory activity while causing no cytotoxicity. The purpose of this study was to examine the impact of IPC extracts on cell proliferation and collagen production in human fibroblasts (CCD-986sk cells). IPC leaves were macerated in 70% and 95% ethanol and the chemical composition of the resulting extracts (IPC70 and IPC95) were determined using high performance liquid chromatography (HPLC). The bioactivity of IPC extracts was examined in CCD-986sk cells, including antioxidant capacity, inhibition of collagenase, effects on cell proliferation and collagen production, as well as wound healing using an in vitro scratch test. Changes in expression of collagen type I (COL1A1), tumor growth factor beta 1 (TGFB1), and beta-fibroblast growth factor (FGF2) genes were also evaluated. The antioxidant and collagenase inhibitory properties of IPC extracts were associated with 3,5-di-caffeoylquinic acid, chlorogenic acid, and ferulic acid. IPC extracts at noncytotoxic concentrations significantly increased cell proliferation, collagen production, and wound healing. These effects appear linked to the upregulation of COL1A1, TGFB1, and FGF2 genes. The bioactivity of the IPC70 extract was greater than that for IPC95. This is useful in cosmeceutical applications for human skin aging. Our findings indicate that IPC extracts have the potential for use in skin anti-aging cosmeceutical preparations.

Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/ß-thalassemia.

BACKGROUND: Hemoglobin E/ß-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of ß-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. METHODS: The phosphoproteome of bone marrow HSCs/CD34⁺ cells from HbE/ß-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺ cells were compared with HbE/ß-thalassemia and normal HSCs. RESULTS: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/ß-thalassemia. CONCLUSIONS: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/ß-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in ß-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/ß-thalassemia.

Additive Effect of a Combination of Artocarpus lakoocha and Glycyrrhiza glabra Extracts on Tyrosinase Inhibition in Melanoma B16 Cells.

Artocarpus lakoocha (Al) and Glycyrrhiza glabra (Gg) extracts have been reported to show tyrosinase inhibitory activity and melanin pigment reduction. This is the first study to assess the combination of Al and Gg extracts in enhancing inhibition of tyrosinase and reduction of melanin pigments. Al and Gg extracted by maceration in 70% and 95% ethanol were analyzed for oxyresveratrol and glabridin using Ultra High Performance Liquid Chromatography. Extracts of Al and Gg singly and combinations of Al95 and Gg95 were tested for cytotoxicity, tyrosinase inhibitory activity, and reduction of melanin pigments in melanoma B16 cells. Al95 had higher antioxidant, tyrosinase inhibitory activity and reduced more melanin pigments in B16 cells compared to Al70, and exhibited higher levels of oxyresveratrol. Gg95 inhibited oxidative stress and mushroom tyrosinase better than Gg70, and exhibited higher levels of glabridin. Combinations of Al95 and Gg95 at various ratios (concentration of 0.1 mg/mL) were not cytotoxic to B16 cells. Interestingly, Al95 and Gg95 combined at a ratio 9:1 reduced melanin pigment up to 53% in B16 cells. This combination of Al95 and Gg95 extracts exhibited the additive effect of reducing melanin pigments by suppressing the expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein-2 (TRP-2) in B16 cells. The combination of Al and Gg extracts could be developed as skin care products for hyperpigmentation treatment.

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein.

BACKGROUND: Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. METHODS AND FINDINGS: Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax-exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax-exposed people reduced P. vivax invasion by up to 54%. CONCLUSIONS: These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.

Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax.

Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy(+) reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14-16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy(+) reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.

Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood.

Plasmodium vivax research has been hampered by the lack of technology for culturing this parasite. Culturing P. vivax is difficult because the parasite selectively invades reticulocytes. Here we describe a modified procedure to establish and maintain short-term cultures of freshly collected P. vivax parasites using reticulocyte-enriched cord blood. Using this method, parasites could be cultured for a month. Manipulation of the culture allowed procurement of synchronized stages of the parasite. This short-term culture method can be easily adapted to study various aspects of the parasite biology.

Triptolide sensitizes resistant cholangiocarcinoma cells to TRAIL-induced apoptosis.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) promotes apoptosis by binding to transmembrane receptors. It is known to induce apoptosis in a wide variety of cancer cells, but TRAIL-resistant cancers have also been documented. In this study, the relative resistance of human cholangiocarcinoma (CCA) cell lines against TRAIL-induced apoptosis is reported and the possible potential synergistic effect with triptolide, a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii, in killing TRAIL-resistant CCA cells is investigated. MATERIALS AND METHODS: Six human CCA cell lines were treated with various concentrations of TRAIL and the resistant cells were identified and subsequently tested for their sensitivity to a combination of TRAIL and triptolide. The susceptibility and resistance of the cells were based on analysis of cytotoxic and apoptotic induction and expression of anti-apoptotic factors (Mcl-1 and cFLIP). RESULTS: The treatment of TRAIL induced a dose-dependent decrease in cell viability in 4 out of the 6 cell lines. A combination of TRAIL and triptolide enhanced cytotoxicity and apoptosis in these 2 resistant cell lines. The combined treatment enhanced activation of caspase-8 and its downstream signaling processes compared with the treatment with either one alone. CONCLUSION: The results presented show that human CCA cells were heterogeneous with respect to susceptibility to TRAIL-induced apoptosis. The combination of TRAIL and triptolide could enhance susceptibility to TRAIL-induced apoptotic killing in these TRAIL-resistant CCA cells, thus offering an alternative approach for the treatment of TRAIL-resistant cholangiocarcinoma.

In vitro production of functional immune cells derived from human hematopoietic stem cells.

Hematopoietic stem cells (HSC) from cord blood are potentially high sources for transplantation due to their low immunogenicity and the presence of the multipotent cells. These cells are capable of differentiating to produce various lineages of blood cells under specific conditions. We have enriched highly purified CD34(+) cells from cord blood, determined in vitro growth of the cells in culture systems in the absence (condition A) or presence of GM-CSF and G-CSF (condition B), and determined the profile of immune cells during the period of cultivation by using flow cytometry. PhytohemagglutininA (PHA) was used as a mitogen to stimulate T lymphocytes derived from hematopoietic stem cells. GM-CSF and G-CSF prolonged the survival of the growing cells and also maintained expansion of cells in blastic stage. By day 12 of cultivation, when cell numbers peaked, various types of immune cells had appeared (CD14(+) cells, CD40(+)HLA-DR(+) cells, CD3(+)CD56(+) cells, CD19(+) cells, CD3(+)CD4(+) cells, CD3(+)CD8(+)cells and CD3-CD56(+)). A significantly higher percentage of monocytes (p = 0.002) were observed under culture with GM-CSF, G-CSF when compared with culture without GM-CSF, G-CSF. In addition, T lymphocytes derived from HSC responded to 50 µg/ml of PHA. This is the first report showing the complete differentiation and proliferation of immune cells derived from CD34(+) HSC under in vitro culture conditions. Lymphocytes, monocytes, dendritic cells and polymorph nuclear cells derived from HSC in vitro are unique, and thus may benefit various studies such as innate immunity and pathophysiology of immune disorders.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the diagnosis of Penicillium marneffei infection.

A monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay was developed for the detection of Penicillium antigen in clinical specimens from patients with Penicillium marneffei infection. The IgM from clone 8C3, reactive with both yeast and mycelial antigens, was immobilized on a microtiter plate. The antigen in serum or urine was captured and detected with biotinylated polyclonal rabbit anti-P. marneffei antibody. The test was sensitive in detecting soluble yeast exoantigen at a concentration as low as 4.88 ng/mL. The reliability of the assay method was evaluated using sera at a dilution of 1:2 from 18 patients with culture-proven penicilliosis, 23 patients with other fungal and bacterial infections, and 125 healthy volunteers. The method exhibited a sensitivity of 72%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 97%, and an accuracy of 97%. However, the sensitivity could be increased to 90% when the specimens were used undiluted. The method was also useful for the detection of antigen secreted in the urine of the patients. The results showed that the newly described assay method can be used in the diagnosis of P. marneffei infection with a high degree of reliability.

Ganoderma lucidum suppresses endothelial cell cytotoxicity and proteinuria in persistent proteinuric focal segmental glomerulosclerosis (FSGS) nephrosis.

A persistent proteinuria is commonly observed in nephrotic patient with focal segmental glomerulosclerosis (FSGS) under treatment with prednisolone+/-cyclophosphamide or with vasodilators (ACEI+AII receptor antagonist, calcium channel blocker and antiplatelet agent). Fourteen such patients with persistent proteinuria were subject to be treated with Ganoderma lucidum. Initial study revealed an enhanced endothelial cell cytotoxicity induced by patient's serum, and an altered immunocirculatory balance with predominant proinflammatory cytokine TNF alpha activity in the presence of defective anti-inflammatory cytokine interleukin-10. Treatment with Ganoderma lucidum suppressed endothelial cell cytotoxicity, restored immunocirculatory balance and successfully suppressed proteinuria in all of these 14 patients.

Glomerular endothelial cytotoxicity and dysfunction in nephrosis with focal segmental glomerulosclerosis.

Glomerular endothelial cell dysfunction (GED) with defective release of vasodilator has been delineated in nephrosis (NS) in vivo and in vitro studies. In NS with focal segmental glomerulosclerosis (FSGS), an immunocirculatory balance may be impaired due to defective anti-inflammatory cytokine. This study aimed at simultaneous determination of both proinflammatory cytokine (tumor necrosis factor alpha) and an anti-inflammatory cytokine (interleukin-10) in NS with FSGS. An endothelial cell cytotoxicity (ECC) was also examined using nephrotic serum. It was shown that (1) the initial endothelial cell cytotoxicity was significantly different from the control, (2) ratio between tumor necrosis alpha and interleukin-10 was significantly elevated, and (3) intrarenal hemodynamics was changed significantly.

Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.

Cholangiocarcinoma is known to be relatively resistant to chemotherapy. One alternative approach is to use a combination of an immunomodulating agent with an anticancer drug. Here we studied the synergistic actions of TNF-alpha and triptolide (a diterpene epoxide prepared from Tripterygium wilfordii), previously shown to have antitumor activity against hamster cholangiocarcinoma (CCA) cells. Three human CCA cell lines (HuCCA-1, HubCCA-1, KKU-100 cell lines) were subjected to a combined treatment of TNF-alpha (0.1-10 ng/ml) and triptolide (5-50 ng/ml) for 24 hours in microculture plates. The combination of TNF-alpha and triptolide had a significantly increased cytotoxic activity over that of triptolide alone (p < 0.05). Under the same conditions, TNF-alpha by itself was not cytotoxic to these cell lines. Similarly, the combined treatment could also accelerate apoptotic cell death in all three human cholangiocarcinoma cell lines. The combined treatment of TNF-alpha at 10 ng/ml and triptolide at 50 ng/ml for 6-10 hours achieved a percentage of apoptotic cells shown by DAPI staining of 18-65%, compared to only 6-20% apoptotic cells for triptolide alone. Analyzing the possible mechanisms of the combined treatment, we found by Western blot that at 6 hours, there was a poly (ADP-ribose) polymerase (PARP) cleavage which was not detectable by the treatment of either TNF-alpha or triptolide alone. The cleavage of PARP was inhibited when the cells were pretreated with the enzyme inhibitor AC-DEVD-CMK, suggesting that apoptosis induced by the combination of TNF-alpha and triptolide involved activation of caspase 3. These results indicate that apoptosis of human cholangiocarcinoma cell lines as induced by a combination of TNF-alpha and triptolide is mediated through caspase 3 activation.

Characterization of inhibitory anti-Duffy binding protein II immunity: approach to Plasmodium vivax vaccine development in Thailand.

Plasmodium vivax Duffy binding protein region II (DBPII) is an important vaccine candidate for antibody-mediated immunity against vivax malaria. A significant challenge for vaccine development of DBPII is its highly polymorphic nature that alters sensitivity to neutralizing antibody responses. Here, we aim to characterize naturally-acquired neutralizing antibodies against DBPII in individual Thai residents to give insight into P. vivax vaccine development in Thailand. Anti-DBPII IgG significantly increased in acute vivax infections compared to uninfected residents and naive controls. Antibody titers and functional anti-DBPII inhibition varied widely and there was no association between titer and inhibition activity. Most high titer plasmas had only a moderate to no functional inhibitory effect on DBP binding to erythrocytes, indicating the protective immunity against DBPII binding is strain specific. Only 5 of 54 samples were highly inhibitory against DBP erythrocyte-binding function. Previously identified target epitopes of inhibitory anti-DBPPII IgG (H1, H2 and H3) were localized to the dimer interface that forms the DARC binding pocket. Amino acid polymorphisms (monomorphic or dimorphic) in H1 and H3 protective epitopes change sensitivity of immune inhibition by alteration of neutralizing antibody recognition. The present study indicates Thai variant H1.T1 (R308S), H3.T1 (D384G) and H3.T3 (K386N) are the most important variants for a DBPII candidate vaccine needed to protect P. vivax in Thai residents.

Glomerular endothelial dysfunction in chronic kidney disease.

A dysfunctioning glomerular endothelium was demonstrated in chronic kidney disease (CKD) patients by means of in vitro endothelial cell cytotoxicity test and of in vivo intrarenal hemodynamic study. An enhanced endothelial cell cytotoxicity in CKD patients was 26.5 +/- 12% as compared to 0.4 +/- 1% of control. An altered intrarenal hemodynamics revealed 1) a reduction in renal plasma flow, 190 +/- 67 mL/min/1.73 m2 versus control 595 +/- 45 mL/min/1.73 m2, and in peritubular capillary flow, 149 +/- 55 mL/min/1.73 m2 versus control 479 +/- 46 mL/min/1.73 m2, 2) an elevated intraglomerular hydrostatic pressure, 55 +/- 2 mmHg versus control 51 mmHg, elevated afferent arteriolar resistance, 13184 dyne x s x cm(-5) versus control 2443 +/- 154 dyne x s x cm(5), and elevated efferent arteriolar resistance, 13591 +/- 7591 dyne x s x cm(-5) versus control 3062 +/- 177 dyne x s x cm(-5). Both enhanced endothelial cell cytotoxicity and altered intrarenal hemodynamics reflect glomerular endothelial dysfunction which is likely responsible for the renal disease progression in CKD.

Proteomic analysis of cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countries, such as Thailand. Distinguishing CCA from hepatocellular carcinoma (HCC) of the liver often requires the use of histochemistry, so molecular markers for diagnosis and prognosis are still required. In this study, the two-dimensional (2-D) protein map of a Thai human bile duct epithelial carcinoma cell line (HuCCA-1) has been compared to human hepatocellular carcinoma cell lines (HepG2 and HCC-S102) and a human breast epithelial cancer cell line (MCF-7). Our results show that HuCCA-1 expressed a unique pattern of proteins. Forty-three major proteins were identified by matching to the map of MCF-7, and by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). Cytokeratins CK8 and CK18 were overexpressed in both HuCCA-1 and HCC, while CK7 and CK19 were only expressed in HuCCA-1. Four specific proteins with MW/pI 57.2/5.21 (U1, vimentin), 42.2/6.20 (U2), 43.2/6.20 (U3, EF-TU), and 42.2/6.40 (U4, unidentified) were absent from HepG2. U2 showed high expression in HuCCA-1, while U1 and U4 showed high expression in HCC-S102. U2 could be separated in 2 proteins, U2/1 (alpha-enolase) and U2/2 (not identified) by using IPG pH 4-7. Galectin-3 showed high expression level in HuCCA-1 by 1-DE immunodetection, and gave only one spot with MW 32.9 kDa and pI 8.29 on 2-DE immunoblotting, Thus, certain proteins, namely CK7, CK19, U2/2 and galectin-3, may be good markers useful for differential diagnosis of cholangiocarcinoma compared to hepatocellular carcinoma.