Independent origin of IFN-alpha and IFN-beta in birds and mammals.

Phylogenetic analysis of type I interferon (IFN) from birds and mammals strongly supported the hypothesis that the gene duplication giving rise to the alpha and beta families of mammalian IFN occurred after the divergence of birds from mammals, whereas the bird IFN that have been designated alpha and beta duplicated independently in the avian lineage. Therefore, IFN designated alpha and beta in birds are not orthologous to those similarly designated in mammals.     

Thrombopoietin production in human hepatic cell cultures (HepG2) is resistant to IFN-alpha, IFN-beta, and IFN-gamma treatment.

Thrombocytopenia is an important complication of interferon (IFN) therapy for chronic viral hepatitis. To study whether IFN interferes with hepatic thrombopoietin (TPO) synthesis, we used the human hepatoma cell line HepG2. Our results show that IFN-alpha, IFN-beta, or IFN-gamma did not impair TPO mRNA expression, as determined by quantitative RT-PCR, even when high IFN doses (up to 5000 U/ml) or long-term incubations (up to 14 days) were applied. Neither was the rate of secretion of immunoreactive TPO reduced on IFN treatment. These findings support the concept that IFNs primarily mediate effects on megakaryocytic cells and platelets rather than on TPO-producing hepatocytes.     

Thermal stability of the WHO international standard of interferon alpha 2b (IFN-alpha 2b): application of new reporter gene assay for IFN-alpha 2b potency determinations

A World Health Organization requirement for biological standards is that they should exhibit long-term stability at their recommended storage temperature. Thermal stability is usually predicted in accelerated thermal degradation studies, where ampoules of the lyophilized standard are stored at elevated temperatures for relatively short times before testing. To confirm the predicted thermal stability of the 2nd international standard of human interferon alpha 2b (IFN-alpha2b; 95/566), we tested the potency of the ampouled contents of this standard after 9 years storage at the customary storage temperature of -20 degrees C in comparison with ampoules of the IS which had been stored continuously at temperatures ranging from -150 degrees C to 56 degrees C. Since IFN-alpha2b potency estimates derived from the results of antiviral assays (AVA) showed high within-assay variability, we investigated a novel reporter gene assay (RGA) based on induction of secreted alkaline phosphatase (SEAP) for comparability and precision of such estimations. We show that this RGA generated comparable estimates with overall lower variation. Additionally, the SEAP conversion of p-nitrophenyl phosphate to yellow product could be followed kinetically. Absorbance readings were shown to increase with time in proportion with increasing concentration of IFN-alpha2b. When the time-dependent increments of absorbance were plotted graphically, the slopes of lines corresponded to concentration. This approach enabled single dilutions of IFN samples, identical in molecular structure to an IFN-alpha2b standard, to be used for potency estimates by interpolation of slope value against those of the standard at fixed concentrations. It appears attractive for high through-put potency testing of various R&D IFN-alpha2b samples.     

Novel controlled-release Lemna-derived IFN-alpha2b (Locteron): pharmacokinetics, pharmacodynamics, and tolerability in a phase I clinical trial.

Locteron, a newly developed controlled-release formulation of Lemna-derived free (unpegylated) recombinant interferon-alpha2b (IFN-alpha2b, Biolex Therapeutics, Pittsboro, NC) in poly(ether-ester) microspheres (PolyActive, OctoPlus N.V., Leiden, the Netherlands), was evaluated in 27 volunteers injected with either 20, 80, or 320 microg Locteron (equivalent to 6.25, 25, or 100 x 10(6) IU, respectively), 80 microg pegylated IFN-alpha2b (PEG-IFN-alpha2b), microspheres not containing IFN-alpha2b, or placebo. Serum free or PEG-IFN-alpha2b and two biomarkers of IFN activity, neopterin and 2',5'-oligoadenylate synthetase (2',5'-OAS), were measured. After injection of 320 microg Locteron, serum IFN-alpha2b remained elevated through 14 days. The elimination half-life of Locteron was more than 2-fold that of PEG-IFN-alpha2b. The effects of 80 microg Locteron and 80 microg PEG-IFN-alpha2b on both neopterin and 2',5'-OAS were in a comparable range. Serum persistence of both these biomarkers was similar at 14 days after 320 microg Locteron compared with 7 days after 80 microg PEG-IFN-alpha2b. Mild, moderate, or severe influenza-like symptoms developed in all 6 subjects receiving 80 microg PEG-IFN-alpha2b. No such symptoms occurred after 20 or 80 microg Locteron doses. Among the 4 recipients of 320 microg Locteron, 1 experienced mild and 2 experienced moderate influenza-like symptoms. Locteron merits further clinical investigation as a hepatitis C therapy suitable for dosing once per 2 weeks.     

Activity of hybrid type I interferons in cells lacking Tyk2: a common region of IFN-alpha 8 induces a response, but IFN-alpha2/8 hybrids can behave like IFN-beta.

Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.     

Plasma homocysteine and immune activation in patients with malignant melanoma undergoing treatment with IFN-alpha.

Immune activation and cell proliferation may contribute to the development of increased homocysteine concentrations in patients with malignant diseases. In this study, we investigated the effect of interferon-alpha (IFN-alpha) on plasma homocysteine concentrations in patients being treated for malignant melanoma. In parallel, neopterin formation and tryptophan degradation were monitored to assess the capacity of IFN-alpha to activate macrophages. Plasma concentrations of homocysteine, folate, and vitamin B(12) were determined in 15 patients with malignant melanoma during 12 weeks of high-dose IFN-alpha therapy. Concurrently, concentrations of neopterin, tryptophan, and kynurenine were measured, and the kynurenine/tryptophan ratio (kyn/trp) was calculated. Homocysteine and folate concentrations during treatment with IFN-alpha did not differ from baseline. In contrast, significant increases in neopterin formation and tryptophan degradation were apparent during IFN-alpha therapy. Plasma concentrations of vitamin B(12) and cysteine also increased. These results indicate that IFN-alpha directly activates macrophages to release neopterin and to degrade tryptophan, but obviously treatment with INF-alpha did not affect homocysteine metabolism.     

Failure to detect antiviral activity in serum and plasma of healthy individuals displaying high activity in ELISA for IFN-alpha and IFN-beta.

The presence of constitutively produced interferon (IFN)-alpha in the blood of healthy individuals has been the subject of contradictory discussions for years. Immunologic as well as biologic test procedures have demonstrated striking differences regarding serum IFN-alpha under physiologic conditions. We investigated the presence of immunoreactive IFN-alpha in serum samples of 923 healthy blood donors by means of a widely used commercially available ELISA. Of these, 254 (27.5%) exhibited detectable serum IFN-alpha levels. The sera of 85.1% of these people also contained IFN-beta. Both IFN were also demonstrated in EDTA-anticoagulated plasma. However, none of these samples exhibited any antiviral effect on human A549 lung carcinoma cells challenged with encephalomyocarditis virus. Samples with high IFN-alpha ELISA activity did not abolish the antiviral action of added natural IFN-alpha, thus excluding IFN-alpha inhibitory factors. The experiments suggest that the detected compounds probably did not represent IFN-alpha but were the result of a cross-reaction with unknown serum components. A variety of disorders has been associated with elevated serum IFN-alpha levels that in most cases were detected by ELISA. In view of our data, these findings need to be carefully reevaluated. For the purpose of monitoring IFN-alpha levels in therapy of atopic, autoimmune, or malignant disorders, an appropriate detection system for IFN-alpha is advisable.     

Measured neutralizing titers of IFN-beta neutralizing antibodies (NAbs) can depend on the preparations of IFN-beta used in the assay.

An immune response to recombinant human protein therapeutics, including type I interferons (IFNs), has the potential to have a serious negative impact on safety and efficacy. Monitoring of patients for neutralizing antibodies (NAbs) often is advisable. In the case of IFN-beta therapy for multiple sclerosis (MS), we obtained reproducible quantitative titers of NAbs using an improved and well-characterized assay based on a 10-fold reduction of a challenge dose of IFN-beta. However, the observed titer was significantly affected by the preparation of IFN-beta used as the assay challenge. NAb titers obtained using IFN-beta1b averaged 3-5-fold lower than titers of the same sample assayed using either IFN-beta1a or human fibroblast-derived IFN-beta. This was the case whether neutralizing serum was obtained from patients on therapy with IFN-beta1a or IFN-beta1b. The reason for this apparent titer difference is not fully understood but appears to be related to protein folding or other structural properties that differentiate the IFN-beta1b both from commercial IFN-beta1a preparations and from human fibroblast-derived IFN-beta.     

LRRK2基因G2019S突变帕金森病相关基因的生物信息学分析

目的 从分子水平揭示富亮氨酸重复激酶2（LRRK2)基因G2019S突变帕金森病的发病机制,为临床诊断及治疗提供新思路。 方法 在公共基因芯片数据库（GEO）中下载LRRK2基因G2019S突变帕金森病的相关基因芯片数据（GSE22491）,其中LRRK2(G2019S)突变帕金森病样本10 例,正常控制组样本8 例,利用Qlucore Omics Explorer（QOE）3.0 软件、DAVID、STRING等在线分析软件对LRRK2基因G2019S突变帕金森病差异基因进行生物信息学分析。结果 QOE3.0分析筛选出1752个LRRK2基因G2019S突变帕金森病差异基因,其中上调191个,下调1561个。对其进行生物信息学分析发现,SKP2、RBX1、SKP1、CUL1、CUL4A 等基因以及核糖体信号通路、氧化磷酸化信号通路、蛋白酶体信号通路、白细胞跨内皮迁移信号通路、磷酸戊糖途径信号通路、枸橼酸信号通路、Fcγ受体（FcγR）介导的吞噬通路等在LRRK2基因G2019S突变帕金森病的发生发展中可能起着重要作用。 结论 通过生物信息学分析LRRK2基因G2019S突变帕金森病相关基因芯片数据,提示LRRK2基因G2019S突变帕金森病发病是多种基因、多种分子机制相互作用的结果,对相关分子机制的进一步分析有利于揭示LRRK2基因G2019S突变帕金森病的发病机制。     

Neutralizing and binding antibodies to IFN-beta: relative frequency in relapsing-remitting multiple sclerosis patients treated with different IFN-beta preparations.

The frequencies of anti-interferon-beta (IFN-beta) antibody development reported to date in patients treated with different IFN-beta preparations are not readily comparable mainly because of differences in underlying diseases and assay methods. Thus, the frequency of neutralizing antibody (NAb) and binding antibody (BAb) development was analyzed in a sample of sera derived from a homogeneous group of relapsing-remitting multiple sclerosis (RRMS) patients treated with different IFN-beta preparations. The frequency of developing NAb and BAb to IFN-beta varied according to the IFN-beta given. Specifically, the NAb seroconversion frequency was significantly higher in patients treated with Betaferon, Schering AG, Berlin, Germany (31.3%) than in patients treated with both preparations of recombinant IFN-beta 1a (Rebif, Serono, Geneva, Switzerland [7.4%] or Avonex, Biogen, Cambridge, MA [6.3%]). Analysis of BAb seroconversion frequency in the same patients revealed that different IFN-beta preparations may also have different capability to induce BAb development and that BAb are produced during IFN-beta therapy at a significantly higher rate than NAb. Our main conclusion is that different human IFN-beta preparations may possess different immunogenicities, leading to varying frequency of development of antibody to IFN-beta in RRMS.     

The combination of IFN-alpha2 and IFN-alpha8 exhibits synergistic antiproliferative activity on renal cell carcinoma (RCC) cell lines through increased binding affinity for IFNAR-2.

Although there are at least 13 interferon-alpha (IFN-alpha) subtypes in humans, interactions between the subtypes remain unknown. To understand IFN-alpha interactions, we examined the antiproliferative activities and the receptor binding affinities of different combinations of IFN-alpha2 and IFN-alpha8 using six renal cell carcinoma (RCC) cell lines. Although IFN-alpha8 was the more potent subtype, synergistic and antagonistic antiproliferative effects were also observed in certain combinations of IFN-alpha2 and IFN-alpha8. To analyze the interactions between IFN-alpha2 and IFN-alpha8, the receptor-binding kinetics of different combinations of IFN-alpha2 and IFN- alpha8 to the IFN-alpha receptors, IFNAR-1 or IFNAR-2, were measured using a surface plasmon resonance-based biosensor. Unexpectedly, the receptor binding kinetics to IFNAR-2 but not to IFNAR-1 were mutually related to antiproliferative activity and increase in the binding speed (K(a)) for IFNAR-2. Moreover, we observed the increased fluorescence intensity (FI) of biotin-labeled IFN-alpha8 to IFNAR-2 by receptor binding inhibition assay with unlabeled IFN-alpha2 but not the other combinations. These findings indicate that the binding manner of IFN-alpha8 for IFNAR-2 is different from that of IFN-alpha2, suggesting that binding of IFN-alpha8 rather than binding of IFN-alpha2 to IFNAR-2 leads to activation and subsequent antiproliferative activity despite the same antiviral activity in RCC.     

Systemic production of IFN-alpha by garlic (Allium sativum) in humans.

The effect of foods on the production of interferon-alpha (IFN-alpha) is currently unknown. Garlic (Allium sativum) used as a folk medicine is reported to stimulate nitric oxide (NO) production. We investigated the systemic increase of NO due to the ingestion of garlic on the plasma IFN-alpha level in normal volunteers. Normal volunteers (10 groups, 10 in each group) ate 2 g fresh garlic, and plasma NO and IFN-alpha levels were determined after 2 and 4 h. The participants were also asked to eat garlic for various periods of time, and plasma NO and IFN-alpha were similarly assayed. Ingestion of 2 g fresh, but not boiled, garlic was found to increase the basal plasma level of NO from 2.7 +/- 0.1 microM to 8.76 +/- 0.21 microM at 2 and 4 h, respectively. The basal plasma IFN-alpha level increased from 9.51 +/- 0.26 nM to 46.3 +/- 1.2 nM in normal volunteers (n = 10) at the same time. The chronic eating of garlic was found to maintain IFN-alpha at high levels for at least 7 days. The exposure of neutrophils to garlic in vivo or in vitro, which also stimulated synthesis of NO in these cells, was found to stimulate IFN-alpha synthesis as measured by the stimulation of IFN-alpha mRNA synthesis. Thus, consumption of garlic resulted in stimulated synthesis of NO and, in turn, IFN-alpha in humans, which could be beneficial in viral or proliferative diseases.     

Ber-H2 (CD30) immunohistochemical staining in malignant melanoma.

Malignant melanoma can be included in the differential diagnosis of Hodgkin's disease, anaplastic large cell lymphoma, or embryonal carcinoma These malignancies express CD30, a marker of diagnostic value. A retrospective immunohistochemical study was undertaken to determine the frequency of immunoreactivity of Ber-H2 (anti-CD30 monoclonal antibody) in malignant melanoma Archival paraffin-embedded tissue from 24 primary and metastatic lesions was used. No Ber-H2 labeling was observed in the majority of the studied cases. Variable weak cytoplasmic staining was present in only one case. The findings are compared with the previous reports claiming frequent CD30 expression in malignant melanoma. We discuss issues pertaining to the interpretation of the Ber-H2 IHC staining in formalin-fixed, paraffin-embedded tissue.     

Immunohistochemical detection of Ki67 in breast cancer correlates with transcriptional regulation of genes related to apoptosis and cell death.

Ki67 is a nuclear protein that is tightly linked to the cell cycle. It is a marker of cell proliferation and has been used to stratify good and poor prognostic categories in invasive breast cancer. Its correlation with gene expression patterns has not been fully elucidated. In this study, Ki67 immunohistochemistry using the MIB-1 antibody was performed on sections cut from 21 formalin-fixed, paraffin-embedded invasive breast cancers. Scoring was determined as nil (no immunostaining), low (10% or less immunopositivity) or high (>10% immunoreactive cells) respectively. The relationship of Ki67 immunohistochemical detection with clinicopathologic parameters was evaluated. Using Affymetrix U133A GeneChips, expression profiles for these tumors were generated and correlated with Ki67 immunohistochemical findings. Analysis of variance was used to define genes that were differentially regulated between the groups. Real-time polymerase chain reaction (PCR) was used to confirm the presence of a downregulated gene. Our results showed high, low and nil Ki67 immunostaining in nine (43%), six (28.5%) and six (28.5%) invasive breast cancers respectively, with increased Ki67 protein expression correlating with high histologic grade (P=0.02), mitotic score (P=0.001) and estrogen receptor immunonegativity (P=0.002). Expression profiling trends of the Ki67 gene mirrored the observed proportions of immunostained cells when the Ki67 immunoscore was >10%. Genes related to apoptosis and cell death (bcl2, MAP2K4, TNF10) were noted to be downregulated in tumors that disclosed >40% Ki67 immunostaining (P<0.001). Downregulation of the bcl2 gene was confirmed at the RNA level by real-time RT-PCR. Differential regulation of these genes, especially bcl2, may contribute to the biological nature of clinically more aggressive and highly proliferative breast cancers.     

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.     

Novel functional biodegradable polymer. III. The construction of poly(gamma-glutamic acid)-sulfonate hydrogel with fibroblast growth factor-2 activity

Poly(gamma-glutamic acid)-sulfonate (gamma-PGA-S) hydrogel, with fibroblast growth factor (FGF)-2 activity was investigated as a novel, next-generation tissue-engineering material. gamma-PGA-net-gamma-PGA-S72 (S72-netgel) was prepared with gamma-PGA-S and gamma-PGA-S72 (72% sulfonated gamma-PGA) to provide the high mobility of gamma-PGA-S72 for FGF-2 activity. Cell adhesion and proliferation activities were evaluated on gamma-PGA and gamma-PGA-S hydrogels along with S72-netgels. Both cell adhesion and proliferation activities of gamma-PGA and gamma-PGA-S hydrogels were low. In contrast, S72-netgels had high cell adhesion and proliferation activities, because of their low swelling ratios and high sulfonic acid group concentrations. Furthermore, S72-netgels had high FGF-2 activity, because gamma-PGA-S72 retained FGF-2 activity when incorporated into S72-netgels. S72-netgels should be useful as next-generation tissue-engineering material containing FGF-2 activity.     

Inherited association of breast and colorectal cancer: limited role of CHEK2 compared with high-penetrance genes

We assessed the association between breast cancer (BC) and colorectal cancer (CRC) from referral pattern to the Regional Genetics Service including molecular analysis. Hospital computer records and/or department referral books were used to identify cases referred to the Regional Genetic Service during a 16-year period (1990-2005 inclusive). All files were reviewed along with associated demographic data, risk assessments, referral details and results from mutation testing. Families were assessed for hereditary breast and colorectal cancer (HBCC) criteria, and all families with eligible individuals were tested for the 1100delC mutation in CHEK2. A total of 8,612 families were identified. One hundred and sixteen of 1,631 (7.5%) families with a primary referral for CRC fulfilled the criteria for HBCC, whereas only 68/6981 (1%) BC referrals fulfilled these criteria. Blood samples were obtained from 113 individuals from 83/184 families. Only 1/113 (1%) has screened positive for the CHEK2 mutation, whereas 14 (17%) families segregate BRCA1/2 mutations and at least 7 (8.5%) carry MLH1/MSH2 mutations. HBCC syndrome, if it exists as a separate entity, is not likely to be due to CHEK2 mutations. Many families are explicable by existing high-penetrance genes, and further work is necessary to elucidate whether the remainder is due to chance or as yet undiscovered genes.