CCL22 mutations in large granular lymphocytic leukemia.

Not available.

Extracellular stimulation of lung fibroblasts with arachidonic acid increases interleukin 11 expression through p38 and ERK signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine that regulates proliferation and motility of cancer cells. Fibroblasts reside in the cancer microenvironment and are the primary source of IL-11. Activated fibroblasts, including cancer-associated fibroblasts that produce IL-11, contribute to the development and progression of cancer, and induce fibrosis associated with cancer. Changes in fatty acid composition or its metabolites, and an increase in free fatty acids have been observed in cancer. The effect of deregulated fatty acids on the development and progression of cancer is not fully understood yet. In the present study, we investigated the effects of fatty acids on mRNA expression and secretion of IL-11 in lung fibroblasts. Among the eight fatty acids added exogenously, arachidonic acid (AA) increased mRNA expression and secretion of IL-11 in lung fibroblasts in a dose-dependent manner. AA-induced upregulation of IL-11 was dependent on the activation of the p38 or ERK MAPK signaling pathways. Furthermore, prostaglandin E2, associated with elevated cyclooxygenase-2 expression, participated in the upregulation of IL-11 via its specific receptor in an autocrine/paracrine manner. These results suggest that AA may mediate IL-11 upregulation in lung fibroblasts in the cancer microenvironment, accompanied by unbalanced fatty acid composition.

Colitis-associated colorectal adenocarcinomas frequently express claudin 18 isoform 2: implications for claudin 18.2 monoclonal antibody therapy.

AIMS: Claudin 18 (CLDN18) is a member of the claudin family of cell surface proteins, which are widely expressed in epithelial cells and play a role in cell-cell adhesion. CLDN18 isoform 2 (CLDN18.2) is specifically expressed in gastric epithelial cells, and is frequently expressed at high levels in gastric adenocarcinoma. On the basis of this, zolbetuximab, a targeted monoclonal antibody, has been developed for patients with CLDN18.2-positive gastro-oesophageal adenocarcinoma. Colitis-associated colorectal adenocarcinomas (CACs) tend to lose intestinal markers and show aberrant gastric mucin expression. Furthermore, clinical trials of human epidermal growth factor receptor 2 (HER2) inhibitor therapy for colorectal carcinoma are ongoing. However, the expression profile of CLDN18.2 and HER2 has not been described in a series of human CACs. METHODS AND RESULTS: We performed immunohistochemistry for CLDN18 and HER2 on 56 consecutive CACs from 55 inflammatory bowel disease patients, and compared the expression profile with that of a control group of 56 sporadic colorectal adenocarcinomas (CRCs). CLDN18.1 expression and CLDN18.2 expression were validated by reverse transcription polymerase chain reaction (PCR) in paraffin-embedded CRC tissues. CLDN18 was positive in 27% (15/56) of CACs and in 5% (3/56) of sporadic CRCs (P = 0.004), and CLDN18-positive CACs were more likely to have lymph node metastasis than CLDN18-negative CACs (67% versus 36%; P = 0.017). CLDN18 expression was significantly associated with MUC5AC expression (P < 0.001) and loss of special AT-rich sequence-binding protein 2 expression (P = 0.005) in CACs. CLDN18.2 was expressed in CRCs that were immunoreactive for CLDN18. Only 4% of CACs were immunoreactive for HER2, and no differences were identified in sporadic CRCs. CONCLUSIONS: These findings suggest that certain CAC cases may be candidates for targeted zolbetuximab therapy.

Osteopontin enhances the migration of lung fibroblasts via upregulation of interleukin-6 through the extracellular signal-regulated kinase (ERK) pathway.

Fibrosis is a phenomenon in which parenchyma is replaced with fibrous tissue. Persistent inflammation accompanied by dysregulation of cytokine production and repeated cycles of inflammation-associated tissue-repair induces fibrosis in various organs including the liver, lung, and kidney. In idiopathic pulmonary fibrosis, production of interleukin (IL)-6 and osteopontin (OPN) are dysregulated. Fibrosis leads to qualitative rather than quantitative changes of fibroblasts at the sites of tissue repair, and this leads to enlargement of fibrotic foci. These fibroblasts are immunohistochemically positive for OPN; however, the effect of overexpressed OPN in fibroblasts is not fully understood yet. In this study, we investigated the effect of OPN on IL-6 secretion and on migration and proliferation of fibroblasts. Lung fibroblasts overexpressing exogenous OPN showed that OPN was linked to the enhancement of cell migration through increased IL-6 secretion via the extracellular signal-regulated kinase (ERK) pathway. These results suggest that OPN may exert its pro-fibrotic functions, such as enhancement of fibroblasts migration by cooperating with chemoattractant IL-6, and may be involved in enlargement of fibrotic foci.

NLRP3 upregulation in A549 cells co-cultured with THP-1 macrophages under hypoxia via deregulated TGF-ß signaling.

NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is one of the key components of the inflammasome. NLRP3 also participates in the regulation of fibrosis independent of the inflammasome. In this study, we analyzed the mechanism of upregulation of NLRP3 expression in A549 cells co-cultured with THP-1 macrophages under hypoxia. Upregulation of NLRP3 was suppressed after treatment with inhibitors of TGF-ß receptor or p38, but not with inhibitors of the IL-1 receptor and SMAD3. The analysis of downstream molecules of TGF-ß signaling in A549 cells co-cultured with THP-1 macrophages under hypoxia showed that TGFBR1 was upregulated and SMAD7 was downregulated. Taken together, these results suggest that the upregulation of NLRP3 in A549 cells is associated with deregulated TGF-ß signaling and that the interaction between NLRP3 and TGF-ß signaling plays a fundamental role in fibrogenesis.

Essential role of PTPN11 mutation in enhanced haematopoietic differentiation potential of induced pluripotent stem cells of juvenile myelomonocytic leukaemia.

We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34+ and CD34+ CD45+ cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34+ cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34+ cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder.

Significance of Molecular Monitoring of T Cells for the Decision of Treatment Strategy for Epstein-Barr Virus-associated Hemophagocytic Lymphohistiocytosis.

A 17-year-old patient with Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis achieved first remission after immunochemotherapy (ICT). However, he had fever with an increase in soluble interleukin-2 receptor, but not in ferritin. Molecular analysis revealed augmented plasma and T-cell EBV loads and reappearance of clonal T cells. Despite achieving second remission, the T-cell EBV load at week 8 after second ICT was almost similar to that at week 8 after first ICT. Hence, cyclosporine was decreased over a 9-month period, with molecular monitoring of plasma and T cells. In this article, we describe how useful molecular monitoring was for detecting relapse and resuming ICT.

Upregulation of osteopontin expression via the interaction of macrophages and fibroblasts under IL-1b stimulation.

BACKGROUND: Fibrosis is attributed to dysregulation of tissue-remodeling. In remodeling areas, fibroblasts and macrophages actively make contact with each other. Osteopontin (OPN) is a pro-fibrotic molecule, whose expression is upregulated by interleukin (IL)-1ß via secretion of its downstream cytokines, such as IL-6. Here, we investigated the effect of interaction between fibroblasts and macrophages under IL-1ß stimulation on the expression of OPN. METHODS: We used human lung fibroblasts and THP-1 macrophages differentiated from THP-1 cells using phorbol 12-myristate 13-acetate. These cells were either cultured alone or co-cultured under IL-1ß stimulation. Secretion of OPN and IL-6 were examined by enzyme-linked immunosorbent assay, and mRNA expression was assessed by quantitative real-time PCR. The effects of siRNA against IL-6 or OPN on OPN expression were evaluated. RESULTS: OPN expression increased when fibroblasts and THP-1 macrophages were co-cultured under IL-1ß stimulation. The siRNA against IL-6 in fibroblasts suppressed the upregulation of OPN expression during co-culture, whereas siRNA against IL-6 in THP-1 macrophages did not. The upregulation of expression of OPN mRNA in fibroblasts or THP-1 macrophages when co-cultured under IL-1ß stimulation was mediated by IL-6 from fibroblasts. OPN from THP-1 macrophages was involved in the increase of OPN expression in fibroblasts. CONCLUSIONS: The present study revealed the crosstalk between fibroblasts and THP-1 macrophages under IL-1ß stimulation, where IL-6 from fibroblasts, stimulated by IL-1ß, upregulated OPN expression in fibroblasts themselves via increase in OPN from THP-1 macrophages. The fibroblasts/macrophages network may induce activation or qualitative changes in both cells, which contributes to inflammation-associated fibrosis.

Panobinostat inhibits the proliferation of CD34+ CD38- cells under stimulation of hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

BACKGROUND: Encouraging responses to histone deacetylase inhibitors have been reported for hematologic malignancies. Here, we report effects of panobinostat and 5-azacytidine on the proliferation of juvenile myelomonocytic leukemia (JMML) CD34+ cells. PROCEDURE: We previously reported that stimulation of JMML CD34+ cells with stem cell factor and thrombopoietin on irradiated murine AGM-S3 cells led to substantial expansion of JMML CD34+ cells that contained leukemic stem cells capable of transplantation into immunodeficient mice. Using this culture system, we evaluated effects of panobinostat and 5-azacytidine on the proliferation of JMML CD34+ cells. RESULTS: Panobinostat dose dependently reduced the numbers of day 7 CD34+ cells generated under stimulation of hematopoietic growth factors on AGM-S3 cells in all eight patients with JMML. These patients possessed various genetic and/or karyotypic abnormalities. CD34+ CD38- cells were substantially more sensitive to panobinostat at 10 and 20 nM than CD34+ CD38+ cells. Panobinostat, however, failed to influence the ability of AGM-S3 cells to stimulate JMML CD34+ cell production. In contrast to HL60 cells, apoptosis and cell cycle arrest in panobinostat-mediated inhibition were at low levels in JMML. The inhibitor also suppressed the factor-dependent proliferation of normal CD34+ cells on AGM-S3 cells. Meanwhile, no substantial inhibitory effects of 5-azacytidine on the growth of JMML CD34+ cells were observed. CONCLUSIONS: These results demonstrate that panobinostat directly suppresses the growth of JMML CD34+ cells, in particular CD34+ CD38- cells, regardless of the genetic abnormality type, suggesting that it is a useful antileukemic drug to target JMML stem cells at a pretransplant stage.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

BACKGROUND: ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. METHODS: We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. RESULTS: Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. CONCLUSION: The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

IL-6 trans-signaling is another pathway to upregulate Osteopontin.

BACKGROUND: Osteopontin (OPN) is a pro-fibrotic molecule upregulated by pro-inflammatory cytokines. Interleukin (IL)-6 functions downstream of IL-1ß and has unique signal pathways: classic- or trans-signaling via membrane-bound IL-6R or soluble IL-6R (sIL-6R). We investigated the effect of IL-6 trans-signaling on the upregulation of OPN. METHODS: We used THP-1 cells and THP-1 macrophages differentiated from THP-1 cells using phorbol 12-myristate 13-acetate (PMA). After IL-1ß stimulation, expression of OPN, IL-6, sIL-6R, and a disintegrin and metalloproteinase 17 (ADAM17) was examined by ELISA and quantitative PCR. The effects of anti-human IL-6 neutralizing antibody, soluble gp130 (sgp130, IL-6 trans-signaling-specific inhibitor), TAPI-1 (ADAM inhibitor) and siRNA against IL-6R or ADAM17 on OPN expression were evaluated. RESULTS: IL-1ß increased OPN and induced IL-6 in THP-1 macrophages. Anti-IL-6 neutralizing antibody and siRNA against IL-6R inhibited OPN upregulation induced by IL-1ß. TAPI-1 significantly inhibited the increase in sIL-6R induced by IL-1ß. Treatment with sgp130 attenuated OPN elevation by IL-1ß, whereas sgp130 did not change OPN levels in THP-1 macrophages without IL-1ß stimulation. ADAM17 was expressed in THP-1 macrophages and THP-1 cells and IL-1ß stimulation significantly increased ADAM17 expression, regardless of PMA treatment. TAPI-1 and siRNA against ADAM17 significantly inhibited OPN increased by IL-1ß. CONCLUSIONS: IL-6 and sIL-6R induced by IL-1ß may trigger IL-6 trans-signaling, contributing to the upregulation of OPN in THP-1 macrophages. Macrophages may be used as a source of IL-6 and sIL-6R and evoke IL-6 trans-signaling.

Acute Myeloid Leukemia in a Patient With X-linked Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is a defect in the differentiation and function of T cells. An increased malignancy risk, mainly lymphatic malignancy, has been described in patients with SCID. We report a patient with X-linked SCID who developed acute myeloid leukemia, derived from the recipient with somatic NRAS mutation 4 months after cord blood transplantation (CBT). Loss of heterozygosity phenomenon of the recipient at 6q14 locus was observed at 2 months post-CBT and progressed to 6q deletion (6q-) chromosome abnormality. Somatic NRAS mutation was detected at 3 months post-CBT. Thus, 6q- and NRAS mutation were strongly associated with the leukemic transformation in our patient.

A Novel Mutation in the Fibrinogen Bß Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia.

We found a novel heterozygous mutation in the fibrinogen Bß chain (c.490G>A) of a 3-year-old girl with congenital hypofibrinogenemia. To clarify the complex genetic mechanism, we made a mini-gene including a FGB c.490G>A mutation region, transfected it into a Chinese Hamster Ovary (CHO) cell line, and analyzed reverse transcription (RT) products. The assembly process and secretion were examined using recombinant mutant fibrinogen. Direct sequencing demonstrated that the mutant RT product was 99 bp longer than the wild-type product, and an extra 99 bases were derived from intron 3. In recombinant expression, a mutant Bß-chain was weakly detected in the transfected CHO cell line, and aberrant fibrinogen was secreted into culture media; however, an aberrant Bß-chain was not detected in plasma. Since the aberrant Bß-chain was catabolized faster in cells, the aberrant Bß-chain in a small amount of secreted fibrinogen may catabolize in the bloodstream. FGB c.490G>A indicated the activation of a cryptic splice site causing the insertion of 99 bp in intron 3. This splicing abnormality led to the production of a Bß-chain possessing 33 aberrant amino acids, including two Cys residues in the coiled-coil domain. Therefore, a splicing abnormality may cause impaired fibrinogen assembly and secretion.

Loss of Mismatched HLA on the Leukemic Blasts of Patients With Relapsed Lymphoid Malignancies Following Bone Marrow Transplantation From Related Donors With HLA Class II Mismatches in the Graft Versus Host Direction.

Mechanisms of relapse of acute lymphoblastic leukemia (ALL) after human leukocyte antigen (HLA) class II mismatched hematopoietic stem cell transplantation (HSCT) remain unclear. We report two children with relapsed ALL after HSCT from related donors with HLA-DRB1 and -DQB1 mismatches in the graft versus host direction. One lost HLA-DRB1, DQB1, and DPB1 alleles, and the other lost one HLA haplotype of the leukemic blasts at relapse. HLA class II loss may be a triggering event for ALL relapse after partially HLA-mismatched-related HSCT. In addition, HLA typing of relapsed leukemic blasts could be vital in the selection of retransplant donors.

Diagnosis and treatment of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder that occurs during infancy and early childhood; this disorder is characterized by hypersensitivity of the myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor in vitro. JMML usually involves somatic and/or germline mutations in the genes of the RAS pathway, including PTPN11, NRAS, KRAS, NF1, and CBL, in the leukemic cells. Almost all patients with JMML experience an aggressive clinical course, and hematopoietic stem cell transplantation (HSCT) is the only curative treatment. A certain proportion of patients with somatic NRAS and germline mutations in CBL, however, have spontaneous resolution. A suitable treatment after diagnosis and conditioning regimen prior to HSCT are yet to be determined, but several clinical trials have been initiated throughout the world to develop suitable pre- or post-allogeneic HSCT treatments and new targeted therapies that are less toxic, to improve patient outcome.

Analysis of KRAS and NRAS Gene Mutations in Arab Asian Children With Acute Leukemia: High Frequency of RAS Mutations in Acute Lymphoblastic Leukemia.

BACKGROUND: KRAS and NRAS gene mutations are frequently observed in childhood leukemia. The objective of this study was to determine the frequency of RAS mutations and the association between RAS mutations and other genetic aberrations in Arab Asian children with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). METHODS: Diagnostic samples of 485 patients (<18 years) with acute leukemia from Iraq and Jordan were obtained, using Flinders Technology Associates filter papers. Polymerase chain reaction and direct sequencing were performed in Japan. RESULTS: RAS mutations were detected in 86/318 (27%) of ALL cases and 35/167 (21%) of AML cases. The frequency of NRAS mutation was similar to that of KRAS mutation in ALL. Two RAS mutations were detected in nine patients. Among 264 Iraqi patients with ALL, RAS mutation was significantly associated with lower initial white blood cell count. Of 57 patients with chimeric transcripts, only two patients with either TEL-AML1 or E2A-PBX1 had KRAS mutation. The frequency of NRAS mutation was four times higher than that of KRAS mutation in AML. FAB-M4 and M5 subsets were associated with RAS mutation. Among 134 Iraqi patients with AML, 18 patients had RAS mutations and other genetic aberrations. In particular, 9 of 25 (36%) with MLL-rearrangement had RAS mutations. CONCLUSION: The prevalence of oncogenic RAS mutations was higher among Arab Asian children than in other countries. RAS mutations in AML were found to coexist with other genetic aberrations, particularly MLL rearrangement.

Incidence of childhood primary immune thrombocytopenic purpura.

There is a discrepancy in the reported incidence of childhood immune thrombocytopenic purpura (ITP) between Europe (2.9-5.3 per 100 000 persons) and Japan (1.91). Ise district is a suitable area in which to conduct epidemiological study because there is little fluctuation in the sociodemographic factors. We performed a retrospective population-based study to clarify the incidence of primary childhood ITP. We calculated person-years for children aged <15 years based on the Ise district demographics between 2002 and 2012. The calculated person-years were 298 533. The number of hospitalized patients in Ise district was 25 (M/F, 14/11) during the study period. The calculated incidence was therefore 8.4 per 100 000 person-years. It is possible that the difference in incidence between the present calculation and that of the European studies is due to variation in accuracy and/or registration criteria between the studies.

STAT3 gene mutations and their association with pure red cell aplasia in large granular lymphocyte leukemia.

Large granular lymphocyte leukemia (LGL L) has been morphologically characterized as a group of lymphoproliferative diseases that include T-cell large granular lymphocytic leukemia (T-LGL L) and chronic lymphoproliferative disorders of natural killer cells (CLPD-NK). We investigated mutations in the Src homology 2 (SH2) domain of the signal transducer and activator of transcription 3 (STAT3) gene in Asian cohorts of T-LGL L and CLPD-NK (n = 42 and 11, respectively). Two mutations, Y640F and D661Y, were identified using direct sequencing or allele-specific (AS) PCR. Y640F and D661Y mutations were found in seven and 18 patients, respectively. Two patients were positive for both mutations. Frequencies of STAT3 mutations in T-LGL L and CLPD-NK were 47.6% and 27.2%, respectively. Pure red cell aplasia (PRCA) was associated with the mutations (P = 0.005). The mutations were persistently found at stable levels in some patients after more than 5 years using AS-quantitative PCR. The results of the present study indicate that the SH2 domain of the STAT3 gene is frequently mutated in Asian T-LGL L and CLPD-NK, and that PRCA is closely correlated with the mutations.

Epithelial-mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions.

Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-ß1 and IL-1ß in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-ß1 inhibitor or IL-1ß neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-ß1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-ß1 in A549 cells and THP-1 macrophages and that of IL-1ß in THP-1 macrophages when each cells were co-cultured. Anti-IL-1ß neutralizing antibody attenuated TGF-ß1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1ß from THP-1 macrophages up-regulated the TGF-ß1 from A549 cells and THP-1 macrophages, and then the TGF-ß1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.

N-homocysteinylation of apolipoprotein A-I impairs the protein's antioxidant ability but not its cholesterol efflux capacity.

A high homocysteine (Hcy) level is a risk factor for atherosclerosis. Hcy can be added to proteins through a process known as N-homocysteinylation. This is thought to be a potential cause of atherosclerosis induction. We previously reported that N-homocysteinylated apolipoprotein A-I (N-Hcy-apoA-I) was identified in normal human plasma. In this study, the effect of N-homocysteinylation on the functions of apoA-I was examined. A kinetic study using dimyristoyl phosphatidylcholine (DMPC) liposomes indicated that N-Hcy-apoA-I showed increased lipid-binding activity compared to wild-type apoA-I. Two reconstituted high-density lipoprotein (rHDL) particles of different sizes (approximately 8.2 nm and 7.6 nm in diameter) were produced by mixing apoA-I and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). However, an increased ratio of large to small particles was found in rHDL prepared with N-Hcy-apoA-I. The normal apoA-I antioxidant ability, estimated by the suppression of conjugated diene formation in low-density lipoprotein (LDL) induced by copper sulfate oxidation, was considerably impaired when using N-Hcy-apoA-I. Although N-Hcy-apoA-I functioned as an oxidant, no significant difference was observed in the cholesterol efflux capacity from THP-1 macrophages between wild-type apoA-I and N-Hcy-apoA-I. These results suggest that N-Hcy-apoA-I might be proatherogenic due to its oxidative behavior but not an attenuation of cholesterol efflux capacity.