Plasma and follicular fluid osteopontin levels during ovarian cycle and their correlation with follicular fluid vascular endothelial growth factor levels.

Osteopontin (OPN) is a multifunctional secreted glycoprotein. We evaluated OPN concentrations in blood and follicular fluid (FF) during the ovarian cycle and their relationship with the production of vascular endothelial growth factor (VEGF), which is involved in the pathophysiology of ovarian hyperstimulation syndrome (OHSS). Twenty-two women undergoing in vitro fertilization (minimal stimulation protocol with clomiphene citrate) were enrolled. Samples were collected (a) on the third day of withdrawal bleeding, (b) 2 days before oocyte retrieval, and (c) on the day of oocyte retrieval. FF was collected during oocyte retrieval. The OPN concentration in each specimen and the VEGF concentration in FF was measured by enzyme-linked immunosorbent assays. Plasma OPN concentrations were (in ng/mL): (a) 416 ± 37.2, (b) 378 ± 35.8, and (c) 390 ± 40.0, with no significant differences between the groups. The OPN concentration in FF was 106 ± 13.4 ng/mL. A positive correlation was found between OPN concentrations in FF and plasma samples. A positive correlation was also found between plasma OPN and FF VEGF concentrations, irrespective of the blood-sampling period. Plasma OPN concentration is suggested to reflect the FF VEGF level at oocyte retrieval and maybe a novel clinical marker for predicting the risk for OHSS.

Immunoproteomic identification of anti-C9 autoimmune antibody in patients with seronegative obstetric antiphospholipid syndrome.

Immunoproteomic analysis was performed to identify unknown, pathology-related molecules in patients with seronegative (SN) obstetric antiphospholipid syndrome (APS) who clinically satisfied the diagnostic criteria for APS, but not the serological criteria. We collected peripheral blood from 13 SN-APS outpatients with known thrombotic predisposition, 13 with no known thrombotic predisposition, and four multiparous women with no history of miscarriage (control). Plasma proteins from volunteers were purified and used as plasma protein antigens. Two-dimensional immunoblotting was performed using pooled control or SN-APS serum samples as the primary antibodies. Mass spectrometry of reactive spots specific to SN-APS serum led to the identification of complement molecule C9. Western blotting using commercial purified alkylated C9 was performed to detect autoantibodies. Examination of individual patient serum identified reactivity in one patient with, and in two patients without known thrombotic predisposition. This study suggests that SN-APS pathologies were associated with autoantibodies that react to specific C9 epitopes.

Diversity of progesterone action on lipopolysaccharide-induced expression changes in cultured human cervical fibroblasts according to inflammation and treatment timing.

PROBLEM: The effectiveness of progesterone (P4) treatment for preventing preterm births is unclear. Its effects on the uterine cervix were tested using cultured human uterine cervical fibroblasts (UCFs). METHOD OF STUDY: UCFs were incubated with lipopolysaccharide (LPS) in the presence or absence of P4 under various conditions. mRNA was subjected to PCR arrays and real-time RT-PCR to assess IL-6, IL-8, IL-1beta, PTGS2, MMP-1, and CXCL10 expression. RESULTS: When exposed to a high-LPS concentration (2.0 µg/mL), expression of these genes was not suppressed by simultaneous P4 (1.0 µmol/L) treatment, but it was significantly inhibited when P4 was administered 1 hour prior to LPS, with the exception of the chemokines IL-8 and CXCL10. Expression of all genes was restricted by P4 under low-level LPS (0.2 µg/mL) stimulation, especially when administered prior to LPS treatment. CONCLUSION: These data suggest that early or prophylactic P4 administration is an effective and important measure for reducing preterm birth risk.

Gonadotropin regulation and role of ovarian osteopontin in the periovulatory period.

Osteopontin (OPN), a secreted glycoprotein, has multiple physiological functions. This study investigated the regulation and roles of OPN in the mouse ovary during the periovulatory stages. Immature female mice were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to simulate follicle maturation and ovulation. In situ hybridization and real-time RT-PCR were performed to assess expression of Opn in the periovulatory ovary. Granulosa cells (GCs) from PMSG-primed immature mice were cultured with or without hCG in the presence or absence of OPN, and effects on expression of Opn, progesterone synthesis, and vascular endothelial growth factor (VEGF) signaling were assessed by real-time RT-PCR, ELISA, and western blotting analysis. Opn transcripts were significantly upregulated 3 h after hCG treatment, followed by a peak at 16 h, and the transcripts localized to GCs. Incubation with hCG significantly increased quantities of Opn transcripts in GCs and OPN levels in the culture medium at 12 and 24 h. Furthermore, OPN treatment caused a significant increase in the levels of Star protein, P 450 cholesterol side-chain cleavage enzyme (p450scc), 3-beta-hydroxysteroid dehydrogenase (Hsd3b), and progesterone in the culture medium. OPN treatment promoted Vegf expression in GCs, which was significantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor. In addition, OPN treatment stimulated phosphorylation of AKT, a downstream PI3K signaling molecule. In conclusion, expression of Opn was upregulated in mouse ovarian GCs in response to a gonadotropin surge through epidermal growth factor receptor signaling, which enhances progesterone synthesis and Vegf expression during the early-luteal phase.

Peroxiredoxin 2 expression is increased in neutrophils of patients with refractory cytopenia with multilineage dysplasia.

Myelodysplastic syndromes (MDS) are heterogeneous clonal disorders characterized by cytopenias that arise due to ineffective haematopoiesis and morphological dysplasia and carry an increased risk of incident acute myeloid leukaemia. The pathogenesis of marrow dysfunction in MDS is multifactorial and consistent with a multistep model and may lead to heterogeneity of MDS. We investigated the proteome profile of circulating neutrophils purified from patients with refractory cytopenia with multilineage dysplasia (RCMD) to identify proteins that have a role in the pathogenesis. Using 2-dimensional difference gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that peroxiredoxin 2 (PRDX2), a member of the peroxiredoxin family that regulates reactive oxygen species, was markedly upregulated in neutrophils of RCMD patients compared to healthy donors. Increased PRDX2 expression in the neutrophils of RCMD patients was confirmed using quantitative reverse transcription polymerase chain reaction, immunoblotting and immunocytochemical analysis. In addition, white blood cell and neutrophil counts in RCMD patients correlated inversely with the PRDX2 expression of. Oxidative stress is a known factor involved in the pathogenesis of MDS, and PRDX2 is associated with tumourigenesis of several solid tumours. Accordingly, our results suggest that PRDX2 may perform an important function in the pathogeneis of RCMD.

Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis.

PURPOSE: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. EXPERIMENTAL DESIGN: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. RESULTS: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. CONCLUSION: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy.

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients.

We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte-related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT-PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP-fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.