CpG ODN的设计、筛选及其对乳腺癌和柯萨奇病毒的作用研究
摘要
CpG ODN是含有非甲基化胞嘧啶-鸟嘌呤二核苷酸基序的单链脱氧寡核苷酸,通过与TLR9的结合,能直接活化机体的天然免疫系统,诱导产生以Th1型为主的免疫应答,间接活化获得性免疫。因此,CpG ODN被广泛的应用于肿瘤、病毒感染及过敏性疾病的治疗中,并在临床前及临床实验研究中取得了可喜的成果。
根据CpG ODN的结构特点,在分析其二级结构的基础上,利用生物信息学方法,设计了一系列含有多个茎环结构的新型CpG ODN,经过筛选,得到了数条分属不同类型的新型CpG ODN。在此基础上,我们对其中的YWODN01和YWODN02进行了深入的研究。结果表明,在小鼠乳腺癌模型中,经引流淋巴结区注射的YWODN 02表现出强效的抑瘤效果,并使相当数量的小鼠发生了肿瘤消退。进一步的研究表明,引流淋巴结及肿瘤内免疫细胞的增多和特异性免疫记忆的调动与YWODN 02的抗肿瘤作用有关。因此,YWODN 02的单独应用可能成为一种对乳腺癌有治疗作用的新型制剂。
此外,YWODN 02在体外能刺激的人PBMC产生抗病毒物质,其培养上清能保护Hela细胞抵抗CVB病毒的感染。在柯萨奇病毒感染小鼠模型中,注射YWODN 02加速了小鼠的病程,加重感染小鼠的心肌的病理改变。这提示,在使用CpG ODN治疗病毒感染性疾病时,应慎重选择应用的时机。
Breast cancer poses a major health risk for women in China and worldwide. Excluding cancers of the skin, it is the most frequently diagnosed cancer and the second leading cause of cancer death in women. Although recent years have seen an improvement in breast cancer detection and a few new therapeutic agents, not much progress has been made in the treatment of the disease. According to the results of a new study, an aggressive, difficult-to-treat form of breast cancer appears to be more common in young African-American and Hispanic women attributed to the effect of social pressure and environmental deterioration. Viral infectious disease is also a major health problem. Among viruses most frequently associated with cases in the U. S., the coxsackieviruses group B (CVB) which infect the myocardium and cause myocarditis and dilated cardiomyopathy, are major etiologic agents. Some research confirmed that the CVB induces the type 1 diabetes. Howecer, up to now, no effective drugs was available in treating cancer or viral infection. So, new types of drugs are badly needed to be developed for contributing the cancer and virus infection.
Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), as an innate immune system agonist through Toll-like receptor 9 (TLR9), have been extensively studied in recent years. Based on functional characteristics, CpG ODNs are divided into A, B and C classes. A-class CpG ODN induces plasmacytoid dendritic cells (pDC) to secret large amount of interferon-α(IFNα) and indirectly activates NK cells and facilitates the induction of CTLs. B-class CpG ODN stimulates the proliferation of B cells and activates them. C-class CpG ODN shares both of the properties of A-class and B-class CpG ODN. These immunstimulatory properties have been attracting the studies on developing CpG ODNs as monotherapeutic agents for the treatment of various cancers and virus infection. In mouse models, administration of B-class CpG ODNs was capable of bringing about rejection of glioblastoma, fibrosarcoma, B16 melanoma, 3LL lung carcinoma, colon carcinoma and breast cancer. In clinical trials, B-class CpG ODNs showed therapeutic potential in patients with recurrent glioblastoma, melanoma and advanced renal cell carcinoma. Also, accumulating clinical studies showed that CpG ODN can efficiently prevent virus infections. A phase I study in health volunteers showed that CpG ODN is safe and well tolerated over a wide dose range, there were also antiviral immune response such as IFN-αhad been detected. So, CpG ODNs manifest very promising potential in clinical applications and can be used as mono-therapeutics to treat cancers or virus infections.
The early empirical structure-activity relationship studies showed that the existence and position of the stem-loop, the length of the stem and the location of CG motifs, in addition to primary structure, was critical for designing novel CpG ODNs’capable of inducing anti-tumor immunity. The stem-loop, the so called structure domain, in addition to provide additional resistant to ubiquitous nucleases, has also been shown to require for the IFN-αinducing properties of CpG ODN,which abolishment of the loop dramatically decreased the IFNαsecretion. However, the position was shown important that stem-loop at the 5’end resulted in loss of activity, which seemed an accessible 5’end is required for the TLR9 recognized. Furthermore, it had been demonstrated that elongated the length of the stem enhanced the IFN-αinductive capacity significantly. In other study, it was found that the active ODNs often have CG dinucleotides in or near the stem loop structure but the inactive ODNs did not. What’s more, point mutations of the CG dinucleotides located within the loop of the ODNs reduce their ability severely. All this proved that the location of CG dinucleotides was important for the ODNs’stimulation.
In the present study, we design novel ODNs based on the secondary structure which is predicted by the DNA‘mfold web server’, and more, there are three principle:1) the open read frame at the 5’end; 2) at least two stem-loop structure; 3) one or more CG within the predicted rigid loop. Follow this general idea, a series of ODNs were developed and screened by using the VSV-protection bioassay and 3H-thymidine incorporation assay. In the process, a batch of novel CpG ODNs were identified: 8 of them were B class CpG ODN, 6 of them were C class CpG ODN. And then, two of them, which named YWODN01 and YWODN02, with the advantage function to evaluate their effects on breast cancer and CVB.
The contents of this paper are focused on the following parts:
1. Designing and screening of novel CpG ODN
In order to develop novel CpG ODN, we have tried to design a series of CpG ODNs that display stem-loops when being analyzed for their secondary structures using‘mfold web server’. In primary screening, proliferation of human PBMC and bioassay for IFN production in human PBMC were used as technical platforms to determine their functions. After the primary designing and screening, the sequences flanking and within the loop were adjusted and predicted again. After multiply predicting and adjusting cycles, up to now, more then 10 of CpG ODN were identified and two of them, which named YWODN01 and YWODN02, belonging to B class CpG ODN and C class CpG ODN respectively, with the advantage function to further analysis.
2. Immunostimulatory properties of YWODN 01 and YWODN 02
2.1 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on human PBMC
Fisrst, to evaluate their heterogeneous, the stimulatory effects of CpG ODN were tested by proliferation assay and VSV protection assay in human PBMC from 6 donors. In human PBMC proliferation assay, both of YWODN 01 and YWODN 02 could stimulate human PBMC to proliferate vigorously. The efficacy was stronger than that induced by 2216 and similar to that induced by 2006 and C274. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate human PBMC to produce efficient IFN with the similar efficacy, shown by 2216 and C274. Dose effect assay showed that YWODN 01 and YWODN 02 at 0.38μgml~(-1) began to induce obvious proliferation, YWODN 01 at 3-12μgml~(-1) induced the proliferative plateau as that induced by 2006 or C274, and that YWODN 02 at 12μg/ml induced the most vigorous proliferation. In VSV protection assay, YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the C274 at 1.5μg/ml or 2216 at 0.75μg/ml, and at dosage of 3~6μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 and YWODN 01 act as C-class and B-class CpG ODN of human use, respectively.
2.2 Activation of immune cells induced by YWODN 01 and YWODN 02
CD69 is an early marker of lymphoid cell activation. To identify types of the cells activated by CpG ODN, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD69 and PE-labeled anti-CD19, CD56, CD4 or CD14 mAbs and then analyzed on a FACS to observe the up-regulation of CD69. The flow cytometry analysis showed that both YWODN 01 and YWODN 02 up-regulated CD69 in CD19+, CD56+ and CD8+ cells, indicating that YWODN 01 and YWODN 02 could activate human B cells, NK cells and T cells. Noticeably, the proportion of CD69+ B and NK cells stimulated with YWODN 02 not YWODN 01 were much higher as compared to that stimulated by control CpG ODNs.
Next, we observed the effect of CpG ODN on costimulatory factor of immune cells.Briefly, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD80, anti-CD86 and anti-HLA-DR mAbs and then analyzed on a FACS. The result showed that YWODN 01 and YWODN 02could upregulate the expression of CD80, CD86 and HLA-DR. All these results suggest that YWODN 01 and YWODN 02 can activate immune cells such as B cells, NK cell and monocytes cells.
2.3 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on mouse splenocytes
Because of the requirement for evaluating the anti-tumor efficacy of YWODN 01 and YWODN 02 in mouse model, we next tested whether they could also activate murine immune cells using proliferation assay, VSV protection assay and CD69-expression assay. In proliferation assay, splenocytes of BALB/c mice were stimulated by YWODN 01, YWODN 02 and control CpG ODN including 1585 (a mouse A-class CpG ODN), 1826 (a mouse B-class CpG ODN) and C274 and then tested for their incorporation of 3H-TdR. The result showed that YWODN 01 and YWODN 02 could stimulate the proliferation of the splenocytes to a level similar to that induced by C274 and 1826 and a much higher level than that induced by 1585 and medium control. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate mouse splenocytes to produce efficient IFN with the similar efficacy, shown by1585 and C274. In CD69-expression assay, mouse splenocytes were stained by fluorescence-labeled mAb after CpG ODN stimulation and analyzed by flow cytometry. The result demonstrated that all CpG ODN used in this assay could increase CD69 expression in CD19+ and panNK+ cells not in CD8+ cells. Comparatively, YWODN 02 was more powerful on activating CD19+ and panNK+ cells than C274 as well as YWODN 01. The results reveal that both of YWODN 01 and YWODN 02 are suitable to be used in mice for evaluating their anti-tumor effect.
2.4 Effect of CG motifs of YWODN 01 on its stimulation of mouse splenocyte
A panel of CpG ODN (YWODN 01 or YWODN 01 with point mutation) were synthesized based on the structure of YWODN 01 and were tested for their capacity to stimulate proliferation of mouse splenocyte. The results showed that point mutations of the CG located within the predicted stem-loop structures of YWODN 01 reduced their ability to activate mouse splenocyte more virogous, which point mutation the CG at the 5’end also inhibited its capacity. These data may indicated the CG with the stem-loop were necessary for its activity.
2.5 The analysis of species-specific of CpG ODN
As reported, the effects of CpG ODN is species-specfic, so we tested the YWODN 01 and YWODN 02 for their capacities to induce the proliferation of splenocyte from rat, cattle, dog, rabbit, mouse and guinea pig and PBMC of swine. Briefly, the splenocyte or PBMC were isolated and stimulated with the indicated CpG ODN (6μg/ml) or PBS for 36h. The cells were tested for their proliferation by 3H-thymidine incorporation assay. These data show that both YWODN 01 and YWODN 02 could stimulate the proliferation of splenocyte from mouse, rat or guinea pig. However, YWODN 02 also showed vigorous stimulation effects on cattle, dog and swine. These findings suggest hat YWODN 02functions in a no specis-spiecfic way.
3. The anti-breast cancer effect of YWODN 01 and YWODN 02
3.1 The direct cytotoxicity of CpG ODN on EMT6
To rule out the possibility that YWODN 02 could directly induce the necrosis of tumor cells, EMT6 cells were stimulated with YWODN 02 in culture for 48 h and then counted by trepan blue staining. The result showed that there were no significant difference in cell numbers between YWODN 02 and PBS treated group. In YWODN 02 (0.1, 1, and 10μg/ml) groups, the cell numbers were 21416±2268; 22083±1233; 23583±2036, respectively. In PBS group, the cell number was 25666±1644. The data suggests that anti-tumor activity of YWODN 02 in mice is not due to its direct cytotoxicity.
3.2 The CpG ODN displayed anti-breast cancer effect in mice
After the in vitro screening, YWODN 01 and YWODN 02 were tested for their in vivo effects on inducing anti-tumor activity in mice inoculated with EMT6 cells, murine breast cancer cell line cells. From day 8 after tumor challenge, tumor-palpated mice were treated with YWODN 01, YWODN 02 or PBS by peritumoral injection for six times in one-day interval. In parallel, mice in a group were s.c. injected with YWODN 02 at tumor-draining lymph node (TDLN) area. The result showed that peritumoral injection of YWODN 01 and YWODN 02 could induce significant inhibition tumor growth. Surprisingly, TDLN administered YWODN 02 displayed the most significant inhibition on tumor growth. Furthermore, intramuscularly and peritoneally administered YWODN 02 was tested for its tumor inhibitory effect in the same model. The result demonstrated that intramuscular and peritoneal injection of YWODN 02 failed to induce significant growth inhibition of the tumors. These data indicate that TDLN-injection is an efficient way for CpG ODN to induce tumor inhibition.
3.3 The anti-breast cance effect of YWODN 01 and YWODN 02
Next, YWODN 01 and YWODN 02 were used to treat mice with palpable tumors from day 8 after tumor challenge by s.c. injection at the right inguinal TDLN area for six times in one-day interval. The result showed that both of YWODN 01 and YWODN 02 could significantly inhibit tumor growth but YWODN 02 showed much stronger effect than YWODN 01. There were 2 mice in YWODN 02-treated group, 9 mice in YWODN 01-treated group and 12 mice in PBS group bearing tumors exceeded 2000 mm3 in size by day 40. Interestingly, developed tumors were completely rejected in 4 out of 12 mice in YWODN 02-treated group. It was also found that both of YWODN 01 and YWODN 02 could prolong the life span of mice challenged with tumor cells, in which YWODN 02 was more significant than YWODN 01. The average survival time was 52 days in PBS treated mice. In contrast, the survival time was 60 days and 80 days in YWODN 01- and YWODN 02-treated mice, respectively. The data indicate that YWODN 02, a C-class CpG ODN, is more vigorous than YWODN 01, a B-class CpG ODN, on inducing anti-breast cancer effect in mice when administered by TDLN-injection.
Based on the observation that TDLN-injection of YWODN 02 induced more potent anti-tumor effect, we further tested the dose-effect of YWODN 02 on tumor inhibition in mice. TDLN-injection of YWODN 02 at 6.25~50μg per mouse could significantly inhibit the growth of EMT6 breast tumor in mice in a dose-dependent manner, whereas YWODN 02 at 3.12μg per mouse didn’t display significant anti-tumor effect. The average survival of mice in PBS group was 54 days, and by day 65 after tumor challenged, 100% of the mice died. In contrast, by day 100 post tumor inoculation, 18%, 50%, 67%, 67% and 83% of the mice injected with YWODN 02 at doses of 3.12, 6.25, 12.5, 25 and 50μg were still alive respectively. The average survivals of mice after treated by YWODN 02 were 66 days in 3.12μg group, 81 days in group and 90, 88 and 94 days in 12.5, 25 and 50μg groups, respectively, indicating that YWODN 02 significantly prolongs the survival of breast cancer bearing mice. This result further confirmed that YWODN 02, as a C-class CpG ODN, can induce effective anti-tumor effect in mice even at a lower dose, such as 6.25μg per mouse.
Upon the result that TDLN-injection of YWODN 02 eradicated the established tumors, we investigated whether YWODN 02 could induce similar inhibition of the tumor developed at distant sites. In one set of experiments, YWODN 02 was injected at either the TDLN area or its counterpart area in the opposite side of tumor. Interestingly, as the tumor sizes were significantly reduced by TDLN injection of YWODN 02, the tumor was also response to the treatment of YWODN 02 at area in the opposite side of tumor. Obviously, the TDLN area-injection of YWODN 02 induced more efficient tumor inhibition. Next, to examine whether TDLN-injection of YWODN 02 were also effective on curing larger tumor, the YWODN 02 therapy was started on day 12 when the average volume of tumors reached to 125 mm3 instead of on day 8 post tumor inoculation. The results showed that mice received TDLN-injection of YWODN 02 from day 12 still showed a significant inhibition in tumor growth, but the efficacy was less compared with that started from day 8.
Next, we observed the effect of prophylactic use of YWODN 02 on tumor inhibition. On day 0, 2, 4, BALB/c mice were s.c. injected with 25μg and 50μg of YWODN 02 or PBS at right inguinal lymph node area. Immediately after the third YWODN 02 injection on day 4, the mice were inoculated with 5×105 EMT6 cells at right back near hind leg. The result showed that although the tumor growth in mice received 25μg of YWODN 02 was delayed compared with that in PBS group, 80% of mice developed tumors and finally died in the group. In contrast, in 50μg YWODN 02 group, only 20% mice developed tumors and 80% mice remained tumor free and were alive for more than 100 days post tumor challenge. On day 40 after tumor challenge, the average tumor weights were 3.92 g in PBS group, 1.48 g in 25μg YWODN 02 group and 0.44 g in 50μg YWODN 02 group, respectively. Together, these findings show that prophylactic injection of YWODN 02 can effectively induce growth inhibition of EMT6 tumor in mice.
3.4 YWODN 02-induced immune responses relevant to anti-breast cancer activity in mice
To understand the effect of YWODN 02 administered through TDLN injection in mice, BALB/c mice with established breast cancer were injected with 25μg of YWODN 02 or PBS as control at TDLN area for six times in one-day interval and sacrificed after 48 h of last injection. The lymph nodes from axillary and inguinal region and spleens were removed for observing their morphologic changes, and analyzing their total cell numbers and cell populations by flow cytometry. YWODN 02-treatment resulted in a significant increase in size of inguinal lymph nodes at injection region and spleens, whereas no obvious change in size of the lymph nodes away from the injection site. In parallel, total cell numbers approximately increased 350%-400% in inguinal lymph nodes at injection region and 100% in spleens in YWODN 02-treated mice compared with those in PBS-treated mice. To the cell populations, there was an average 4-fold increase in the number of CD4 or CD8 T cells, 15-fold increase in the number of CD11c DCs, and 20-fold increase in NK cells obtained from inguinal lymph nodes at injection region of YWODN 02-treated mice compared to those in PBS-treated mice. Thus, TDLN injection of YWODN 02 is likely to recruit and expand T cells, NK cells and DC in local lymph nodes, accompanying a systemic splenomegaly, which may all be involved in the anti-tumor activity induced by YWODN 02.
To study the effects of YWODN 02 on tumor and infiltration of immune cells in the tumor, mice with established EMT6 tumors were treated with YWODN 02 or PBS through TDLN injection for six times in one-day interval. After 48 h of the last injection, tumors were removed for histological study by H-E staining and immune cell counting by flow cytometry. The result showed that a large area of necrosis located in periphery of tumors was observed in YWODN 02-treated mice but not in PBS-treated mice. In PBS-treated mice, tumor with well demarcation revealed complete cell shape with clear cytoplasm and typical nuclei. In contrast, the cell shape was completely destroyed in necrotic tumor tissue derived from YWODN 02-treated mice. To understand the reason of tumor necrosis in YWODN 02-treated mice, immune cells in tumor homogenates were counted by flow cytometry. The percentage of CD4+, CD8+, CD11c+ and NK cells in tumor homogenates from YWODN 02-treated mice were increased to 4-fold, 9-fold, 1.6-fold and 2-fold, respectively, compared with those from PBS-treated mice. It seemed that TDLN-injection of YWODN 02 triggered the migration of T cells, NK cells as well as DCs into tumor and the cells somehow mediated the growth inhibition and necrosis of tumor cells.
To confirm whether or not YWODN 02 could also induce specific immune response in mice through TDLN injection, we selected 13 tumor-free mice from three separate experiments, which had undergone the first-round of YWODN 02 treatment from day 8 post-EMT6 cell inoculations, as models after living more than 100 days for observing specific anti-tumor immune response. The mice were divided into two groups and re-challenged with EMT6 cells (5×105 per mouse) same as that in the first challenged or CT26 colon carcinoma cells (2×105 per mouse), respectively, at right back near hind leg without YWODN 02 treatment. The tumor development was monitored further on. The result showed that no tumor was developed in 7 mice re-challenged with EMT6 cells but there were tumors developed in 5 of 6 mice re-challenged with CT26, indicating the YWODN 02 treatment could facilitate the tumor bearing mice to broke the immune tolerance to tumor cells and consequently establish tumor specific immune memory that is sufficient to protect the mice from re-challenge of the same tumor. Promisingly, this property of YWODN 02 might benefit tumor treatment in clinic setting, either in tumor bearing patients or in the patients received tumor removing operation.
4. The effect of YWODN02 on CVB
The previous work of our laboratory showed that BW001 could induce human PBMC to produce anti-VSV, CVB and anti-SARS-CoV activity. First, a CVB/Hela protection assay was used to test its effect on CVB in vitro. Next, a mouse model was used to evaluate its effect on CVB in vivo.
4.1 The effect of YWODN02 on CVB in vitro
To further investigate whether YWODN 02 could stimulate PBMC to produce anti-CVB activity, human PBMC were stimulated by CpG ODN for 36 h and the supernants were collected. And then, Hela cells were treated with the supernatants, followed by attacking with 10×TCID50 of CVB3. The result showed that YWODN 02 stimulated supernatant could significantly protect Hela cells from CVB3 infection.
Dose effect assay showed that YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the 2216 at 0.75μg/ml, and at dosage of 3~6μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 could stimulate PBMC to produce anti-CVB activity.
4.2 The effect of YWODN02 on CVB-infected mice
To test its anti-CVB effect in vitro, female Blab/c mice were infected with CVB3 (4×100 TCID50/0.2 mL) by intraperitoneal (i.p.) injection. From 1 hour after virus challenge, mice were treated with YWODN 02 or PBS by i.p. injection for six times in succession. Healthy control mice remained untreated. Body weight was monitored over a period of 9 days and at day 10, the mice were sacrificed for removing heart and heart tissue was histological evaluated by H-E staining. However, in vivo study showed that YWODN 02 reduced the body weight of CVB3-infected mice and aggravated the pathological changes in the myocardium.
Taken together, based on their secondary structure, we designed and screened 50 CpG ODN with novel structures and successfully identified a batch of potent CpG ODN, including 10 B-class CpG ODNs or C-class CpG ODNs. The novel CpG ODN could be used as adjuvant for currently used vaccines and as monotherapeutics to treat cancer and infectious diseases.Among the novel CpG ODN we discovered, two of them, designated as YWODN 01 and YWODN 02 respectively, have been studied for their anti-tumor activities in mice inoculated with EMT6 breast cancer cells. We have found that YWODN 02, identified as a C-class CpG-ODN, displays more potent anti-tumor activity than YWODN01, identified as a B-class CpG-ODN. Inspiringly, after the tumor establishment, YWODN 02 injected through TDLN area induces dramatic retardation of tumor growth and complete tumor regression. This result suggests that YWODN 02, a C-class CpG ODN, may have the potential to be developed as a monotherapeutic medicament for the treatment of breast cancer through TDLN-injection.The data of CpG ODN in CVB-infected mice may indicated that when used for the treatment of viral-infected diseases in vivo, CpG ODN may function in a complicated way. These data indicated that its administration time, route and dosage, should be taken into full consideration.
根据CpG ODN的结构特点,在分析其二级结构的基础上,利用生物信息学方法,设计了一系列含有多个茎环结构的新型CpG ODN,经过筛选,得到了数条分属不同类型的新型CpG ODN。在此基础上,我们对其中的YWODN01和YWODN02进行了深入的研究。结果表明,在小鼠乳腺癌模型中,经引流淋巴结区注射的YWODN 02表现出强效的抑瘤效果,并使相当数量的小鼠发生了肿瘤消退。进一步的研究表明,引流淋巴结及肿瘤内免疫细胞的增多和特异性免疫记忆的调动与YWODN 02的抗肿瘤作用有关。因此,YWODN 02的单独应用可能成为一种对乳腺癌有治疗作用的新型制剂。
此外,YWODN 02在体外能刺激的人PBMC产生抗病毒物质,其培养上清能保护Hela细胞抵抗CVB病毒的感染。在柯萨奇病毒感染小鼠模型中,注射YWODN 02加速了小鼠的病程,加重感染小鼠的心肌的病理改变。这提示,在使用CpG ODN治疗病毒感染性疾病时,应慎重选择应用的时机。
Breast cancer poses a major health risk for women in China and worldwide. Excluding cancers of the skin, it is the most frequently diagnosed cancer and the second leading cause of cancer death in women. Although recent years have seen an improvement in breast cancer detection and a few new therapeutic agents, not much progress has been made in the treatment of the disease. According to the results of a new study, an aggressive, difficult-to-treat form of breast cancer appears to be more common in young African-American and Hispanic women attributed to the effect of social pressure and environmental deterioration. Viral infectious disease is also a major health problem. Among viruses most frequently associated with cases in the U. S., the coxsackieviruses group B (CVB) which infect the myocardium and cause myocarditis and dilated cardiomyopathy, are major etiologic agents. Some research confirmed that the CVB induces the type 1 diabetes. Howecer, up to now, no effective drugs was available in treating cancer or viral infection. So, new types of drugs are badly needed to be developed for contributing the cancer and virus infection.
Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), as an innate immune system agonist through Toll-like receptor 9 (TLR9), have been extensively studied in recent years. Based on functional characteristics, CpG ODNs are divided into A, B and C classes. A-class CpG ODN induces plasmacytoid dendritic cells (pDC) to secret large amount of interferon-α(IFNα) and indirectly activates NK cells and facilitates the induction of CTLs. B-class CpG ODN stimulates the proliferation of B cells and activates them. C-class CpG ODN shares both of the properties of A-class and B-class CpG ODN. These immunstimulatory properties have been attracting the studies on developing CpG ODNs as monotherapeutic agents for the treatment of various cancers and virus infection. In mouse models, administration of B-class CpG ODNs was capable of bringing about rejection of glioblastoma, fibrosarcoma, B16 melanoma, 3LL lung carcinoma, colon carcinoma and breast cancer. In clinical trials, B-class CpG ODNs showed therapeutic potential in patients with recurrent glioblastoma, melanoma and advanced renal cell carcinoma. Also, accumulating clinical studies showed that CpG ODN can efficiently prevent virus infections. A phase I study in health volunteers showed that CpG ODN is safe and well tolerated over a wide dose range, there were also antiviral immune response such as IFN-αhad been detected. So, CpG ODNs manifest very promising potential in clinical applications and can be used as mono-therapeutics to treat cancers or virus infections.
The early empirical structure-activity relationship studies showed that the existence and position of the stem-loop, the length of the stem and the location of CG motifs, in addition to primary structure, was critical for designing novel CpG ODNs’capable of inducing anti-tumor immunity. The stem-loop, the so called structure domain, in addition to provide additional resistant to ubiquitous nucleases, has also been shown to require for the IFN-αinducing properties of CpG ODN,which abolishment of the loop dramatically decreased the IFNαsecretion. However, the position was shown important that stem-loop at the 5’end resulted in loss of activity, which seemed an accessible 5’end is required for the TLR9 recognized. Furthermore, it had been demonstrated that elongated the length of the stem enhanced the IFN-αinductive capacity significantly. In other study, it was found that the active ODNs often have CG dinucleotides in or near the stem loop structure but the inactive ODNs did not. What’s more, point mutations of the CG dinucleotides located within the loop of the ODNs reduce their ability severely. All this proved that the location of CG dinucleotides was important for the ODNs’stimulation.
In the present study, we design novel ODNs based on the secondary structure which is predicted by the DNA‘mfold web server’, and more, there are three principle:1) the open read frame at the 5’end; 2) at least two stem-loop structure; 3) one or more CG within the predicted rigid loop. Follow this general idea, a series of ODNs were developed and screened by using the VSV-protection bioassay and 3H-thymidine incorporation assay. In the process, a batch of novel CpG ODNs were identified: 8 of them were B class CpG ODN, 6 of them were C class CpG ODN. And then, two of them, which named YWODN01 and YWODN02, with the advantage function to evaluate their effects on breast cancer and CVB.
The contents of this paper are focused on the following parts:
1. Designing and screening of novel CpG ODN
In order to develop novel CpG ODN, we have tried to design a series of CpG ODNs that display stem-loops when being analyzed for their secondary structures using‘mfold web server’. In primary screening, proliferation of human PBMC and bioassay for IFN production in human PBMC were used as technical platforms to determine their functions. After the primary designing and screening, the sequences flanking and within the loop were adjusted and predicted again. After multiply predicting and adjusting cycles, up to now, more then 10 of CpG ODN were identified and two of them, which named YWODN01 and YWODN02, belonging to B class CpG ODN and C class CpG ODN respectively, with the advantage function to further analysis.
2. Immunostimulatory properties of YWODN 01 and YWODN 02
2.1 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on human PBMC
Fisrst, to evaluate their heterogeneous, the stimulatory effects of CpG ODN were tested by proliferation assay and VSV protection assay in human PBMC from 6 donors. In human PBMC proliferation assay, both of YWODN 01 and YWODN 02 could stimulate human PBMC to proliferate vigorously. The efficacy was stronger than that induced by 2216 and similar to that induced by 2006 and C274. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate human PBMC to produce efficient IFN with the similar efficacy, shown by 2216 and C274. Dose effect assay showed that YWODN 01 and YWODN 02 at 0.38μgml~(-1) began to induce obvious proliferation, YWODN 01 at 3-12μgml~(-1) induced the proliferative plateau as that induced by 2006 or C274, and that YWODN 02 at 12μg/ml induced the most vigorous proliferation. In VSV protection assay, YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the C274 at 1.5μg/ml or 2216 at 0.75μg/ml, and at dosage of 3~6μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 and YWODN 01 act as C-class and B-class CpG ODN of human use, respectively.
2.2 Activation of immune cells induced by YWODN 01 and YWODN 02
CD69 is an early marker of lymphoid cell activation. To identify types of the cells activated by CpG ODN, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD69 and PE-labeled anti-CD19, CD56, CD4 or CD14 mAbs and then analyzed on a FACS to observe the up-regulation of CD69. The flow cytometry analysis showed that both YWODN 01 and YWODN 02 up-regulated CD69 in CD19+, CD56+ and CD8+ cells, indicating that YWODN 01 and YWODN 02 could activate human B cells, NK cells and T cells. Noticeably, the proportion of CD69+ B and NK cells stimulated with YWODN 02 not YWODN 01 were much higher as compared to that stimulated by control CpG ODNs.
Next, we observed the effect of CpG ODN on costimulatory factor of immune cells.Briefly, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD80, anti-CD86 and anti-HLA-DR mAbs and then analyzed on a FACS. The result showed that YWODN 01 and YWODN 02could upregulate the expression of CD80, CD86 and HLA-DR. All these results suggest that YWODN 01 and YWODN 02 can activate immune cells such as B cells, NK cell and monocytes cells.
2.3 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on mouse splenocytes
Because of the requirement for evaluating the anti-tumor efficacy of YWODN 01 and YWODN 02 in mouse model, we next tested whether they could also activate murine immune cells using proliferation assay, VSV protection assay and CD69-expression assay. In proliferation assay, splenocytes of BALB/c mice were stimulated by YWODN 01, YWODN 02 and control CpG ODN including 1585 (a mouse A-class CpG ODN), 1826 (a mouse B-class CpG ODN) and C274 and then tested for their incorporation of 3H-TdR. The result showed that YWODN 01 and YWODN 02 could stimulate the proliferation of the splenocytes to a level similar to that induced by C274 and 1826 and a much higher level than that induced by 1585 and medium control. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate mouse splenocytes to produce efficient IFN with the similar efficacy, shown by1585 and C274. In CD69-expression assay, mouse splenocytes were stained by fluorescence-labeled mAb after CpG ODN stimulation and analyzed by flow cytometry. The result demonstrated that all CpG ODN used in this assay could increase CD69 expression in CD19+ and panNK+ cells not in CD8+ cells. Comparatively, YWODN 02 was more powerful on activating CD19+ and panNK+ cells than C274 as well as YWODN 01. The results reveal that both of YWODN 01 and YWODN 02 are suitable to be used in mice for evaluating their anti-tumor effect.
2.4 Effect of CG motifs of YWODN 01 on its stimulation of mouse splenocyte
A panel of CpG ODN (YWODN 01 or YWODN 01 with point mutation) were synthesized based on the structure of YWODN 01 and were tested for their capacity to stimulate proliferation of mouse splenocyte. The results showed that point mutations of the CG located within the predicted stem-loop structures of YWODN 01 reduced their ability to activate mouse splenocyte more virogous, which point mutation the CG at the 5’end also inhibited its capacity. These data may indicated the CG with the stem-loop were necessary for its activity.
2.5 The analysis of species-specific of CpG ODN
As reported, the effects of CpG ODN is species-specfic, so we tested the YWODN 01 and YWODN 02 for their capacities to induce the proliferation of splenocyte from rat, cattle, dog, rabbit, mouse and guinea pig and PBMC of swine. Briefly, the splenocyte or PBMC were isolated and stimulated with the indicated CpG ODN (6μg/ml) or PBS for 36h. The cells were tested for their proliferation by 3H-thymidine incorporation assay. These data show that both YWODN 01 and YWODN 02 could stimulate the proliferation of splenocyte from mouse, rat or guinea pig. However, YWODN 02 also showed vigorous stimulation effects on cattle, dog and swine. These findings suggest hat YWODN 02functions in a no specis-spiecfic way.
3. The anti-breast cancer effect of YWODN 01 and YWODN 02
3.1 The direct cytotoxicity of CpG ODN on EMT6
To rule out the possibility that YWODN 02 could directly induce the necrosis of tumor cells, EMT6 cells were stimulated with YWODN 02 in culture for 48 h and then counted by trepan blue staining. The result showed that there were no significant difference in cell numbers between YWODN 02 and PBS treated group. In YWODN 02 (0.1, 1, and 10μg/ml) groups, the cell numbers were 21416±2268; 22083±1233; 23583±2036, respectively. In PBS group, the cell number was 25666±1644. The data suggests that anti-tumor activity of YWODN 02 in mice is not due to its direct cytotoxicity.
3.2 The CpG ODN displayed anti-breast cancer effect in mice
After the in vitro screening, YWODN 01 and YWODN 02 were tested for their in vivo effects on inducing anti-tumor activity in mice inoculated with EMT6 cells, murine breast cancer cell line cells. From day 8 after tumor challenge, tumor-palpated mice were treated with YWODN 01, YWODN 02 or PBS by peritumoral injection for six times in one-day interval. In parallel, mice in a group were s.c. injected with YWODN 02 at tumor-draining lymph node (TDLN) area. The result showed that peritumoral injection of YWODN 01 and YWODN 02 could induce significant inhibition tumor growth. Surprisingly, TDLN administered YWODN 02 displayed the most significant inhibition on tumor growth. Furthermore, intramuscularly and peritoneally administered YWODN 02 was tested for its tumor inhibitory effect in the same model. The result demonstrated that intramuscular and peritoneal injection of YWODN 02 failed to induce significant growth inhibition of the tumors. These data indicate that TDLN-injection is an efficient way for CpG ODN to induce tumor inhibition.
3.3 The anti-breast cance effect of YWODN 01 and YWODN 02
Next, YWODN 01 and YWODN 02 were used to treat mice with palpable tumors from day 8 after tumor challenge by s.c. injection at the right inguinal TDLN area for six times in one-day interval. The result showed that both of YWODN 01 and YWODN 02 could significantly inhibit tumor growth but YWODN 02 showed much stronger effect than YWODN 01. There were 2 mice in YWODN 02-treated group, 9 mice in YWODN 01-treated group and 12 mice in PBS group bearing tumors exceeded 2000 mm3 in size by day 40. Interestingly, developed tumors were completely rejected in 4 out of 12 mice in YWODN 02-treated group. It was also found that both of YWODN 01 and YWODN 02 could prolong the life span of mice challenged with tumor cells, in which YWODN 02 was more significant than YWODN 01. The average survival time was 52 days in PBS treated mice. In contrast, the survival time was 60 days and 80 days in YWODN 01- and YWODN 02-treated mice, respectively. The data indicate that YWODN 02, a C-class CpG ODN, is more vigorous than YWODN 01, a B-class CpG ODN, on inducing anti-breast cancer effect in mice when administered by TDLN-injection.
Based on the observation that TDLN-injection of YWODN 02 induced more potent anti-tumor effect, we further tested the dose-effect of YWODN 02 on tumor inhibition in mice. TDLN-injection of YWODN 02 at 6.25~50μg per mouse could significantly inhibit the growth of EMT6 breast tumor in mice in a dose-dependent manner, whereas YWODN 02 at 3.12μg per mouse didn’t display significant anti-tumor effect. The average survival of mice in PBS group was 54 days, and by day 65 after tumor challenged, 100% of the mice died. In contrast, by day 100 post tumor inoculation, 18%, 50%, 67%, 67% and 83% of the mice injected with YWODN 02 at doses of 3.12, 6.25, 12.5, 25 and 50μg were still alive respectively. The average survivals of mice after treated by YWODN 02 were 66 days in 3.12μg group, 81 days in group and 90, 88 and 94 days in 12.5, 25 and 50μg groups, respectively, indicating that YWODN 02 significantly prolongs the survival of breast cancer bearing mice. This result further confirmed that YWODN 02, as a C-class CpG ODN, can induce effective anti-tumor effect in mice even at a lower dose, such as 6.25μg per mouse.
Upon the result that TDLN-injection of YWODN 02 eradicated the established tumors, we investigated whether YWODN 02 could induce similar inhibition of the tumor developed at distant sites. In one set of experiments, YWODN 02 was injected at either the TDLN area or its counterpart area in the opposite side of tumor. Interestingly, as the tumor sizes were significantly reduced by TDLN injection of YWODN 02, the tumor was also response to the treatment of YWODN 02 at area in the opposite side of tumor. Obviously, the TDLN area-injection of YWODN 02 induced more efficient tumor inhibition. Next, to examine whether TDLN-injection of YWODN 02 were also effective on curing larger tumor, the YWODN 02 therapy was started on day 12 when the average volume of tumors reached to 125 mm3 instead of on day 8 post tumor inoculation. The results showed that mice received TDLN-injection of YWODN 02 from day 12 still showed a significant inhibition in tumor growth, but the efficacy was less compared with that started from day 8.
Next, we observed the effect of prophylactic use of YWODN 02 on tumor inhibition. On day 0, 2, 4, BALB/c mice were s.c. injected with 25μg and 50μg of YWODN 02 or PBS at right inguinal lymph node area. Immediately after the third YWODN 02 injection on day 4, the mice were inoculated with 5×105 EMT6 cells at right back near hind leg. The result showed that although the tumor growth in mice received 25μg of YWODN 02 was delayed compared with that in PBS group, 80% of mice developed tumors and finally died in the group. In contrast, in 50μg YWODN 02 group, only 20% mice developed tumors and 80% mice remained tumor free and were alive for more than 100 days post tumor challenge. On day 40 after tumor challenge, the average tumor weights were 3.92 g in PBS group, 1.48 g in 25μg YWODN 02 group and 0.44 g in 50μg YWODN 02 group, respectively. Together, these findings show that prophylactic injection of YWODN 02 can effectively induce growth inhibition of EMT6 tumor in mice.
3.4 YWODN 02-induced immune responses relevant to anti-breast cancer activity in mice
To understand the effect of YWODN 02 administered through TDLN injection in mice, BALB/c mice with established breast cancer were injected with 25μg of YWODN 02 or PBS as control at TDLN area for six times in one-day interval and sacrificed after 48 h of last injection. The lymph nodes from axillary and inguinal region and spleens were removed for observing their morphologic changes, and analyzing their total cell numbers and cell populations by flow cytometry. YWODN 02-treatment resulted in a significant increase in size of inguinal lymph nodes at injection region and spleens, whereas no obvious change in size of the lymph nodes away from the injection site. In parallel, total cell numbers approximately increased 350%-400% in inguinal lymph nodes at injection region and 100% in spleens in YWODN 02-treated mice compared with those in PBS-treated mice. To the cell populations, there was an average 4-fold increase in the number of CD4 or CD8 T cells, 15-fold increase in the number of CD11c DCs, and 20-fold increase in NK cells obtained from inguinal lymph nodes at injection region of YWODN 02-treated mice compared to those in PBS-treated mice. Thus, TDLN injection of YWODN 02 is likely to recruit and expand T cells, NK cells and DC in local lymph nodes, accompanying a systemic splenomegaly, which may all be involved in the anti-tumor activity induced by YWODN 02.
To study the effects of YWODN 02 on tumor and infiltration of immune cells in the tumor, mice with established EMT6 tumors were treated with YWODN 02 or PBS through TDLN injection for six times in one-day interval. After 48 h of the last injection, tumors were removed for histological study by H-E staining and immune cell counting by flow cytometry. The result showed that a large area of necrosis located in periphery of tumors was observed in YWODN 02-treated mice but not in PBS-treated mice. In PBS-treated mice, tumor with well demarcation revealed complete cell shape with clear cytoplasm and typical nuclei. In contrast, the cell shape was completely destroyed in necrotic tumor tissue derived from YWODN 02-treated mice. To understand the reason of tumor necrosis in YWODN 02-treated mice, immune cells in tumor homogenates were counted by flow cytometry. The percentage of CD4+, CD8+, CD11c+ and NK cells in tumor homogenates from YWODN 02-treated mice were increased to 4-fold, 9-fold, 1.6-fold and 2-fold, respectively, compared with those from PBS-treated mice. It seemed that TDLN-injection of YWODN 02 triggered the migration of T cells, NK cells as well as DCs into tumor and the cells somehow mediated the growth inhibition and necrosis of tumor cells.
To confirm whether or not YWODN 02 could also induce specific immune response in mice through TDLN injection, we selected 13 tumor-free mice from three separate experiments, which had undergone the first-round of YWODN 02 treatment from day 8 post-EMT6 cell inoculations, as models after living more than 100 days for observing specific anti-tumor immune response. The mice were divided into two groups and re-challenged with EMT6 cells (5×105 per mouse) same as that in the first challenged or CT26 colon carcinoma cells (2×105 per mouse), respectively, at right back near hind leg without YWODN 02 treatment. The tumor development was monitored further on. The result showed that no tumor was developed in 7 mice re-challenged with EMT6 cells but there were tumors developed in 5 of 6 mice re-challenged with CT26, indicating the YWODN 02 treatment could facilitate the tumor bearing mice to broke the immune tolerance to tumor cells and consequently establish tumor specific immune memory that is sufficient to protect the mice from re-challenge of the same tumor. Promisingly, this property of YWODN 02 might benefit tumor treatment in clinic setting, either in tumor bearing patients or in the patients received tumor removing operation.
4. The effect of YWODN02 on CVB
The previous work of our laboratory showed that BW001 could induce human PBMC to produce anti-VSV, CVB and anti-SARS-CoV activity. First, a CVB/Hela protection assay was used to test its effect on CVB in vitro. Next, a mouse model was used to evaluate its effect on CVB in vivo.
4.1 The effect of YWODN02 on CVB in vitro
To further investigate whether YWODN 02 could stimulate PBMC to produce anti-CVB activity, human PBMC were stimulated by CpG ODN for 36 h and the supernants were collected. And then, Hela cells were treated with the supernatants, followed by attacking with 10×TCID50 of CVB3. The result showed that YWODN 02 stimulated supernatant could significantly protect Hela cells from CVB3 infection.
Dose effect assay showed that YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the 2216 at 0.75μg/ml, and at dosage of 3~6μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 could stimulate PBMC to produce anti-CVB activity.
4.2 The effect of YWODN02 on CVB-infected mice
To test its anti-CVB effect in vitro, female Blab/c mice were infected with CVB3 (4×100 TCID50/0.2 mL) by intraperitoneal (i.p.) injection. From 1 hour after virus challenge, mice were treated with YWODN 02 or PBS by i.p. injection for six times in succession. Healthy control mice remained untreated. Body weight was monitored over a period of 9 days and at day 10, the mice were sacrificed for removing heart and heart tissue was histological evaluated by H-E staining. However, in vivo study showed that YWODN 02 reduced the body weight of CVB3-infected mice and aggravated the pathological changes in the myocardium.
Taken together, based on their secondary structure, we designed and screened 50 CpG ODN with novel structures and successfully identified a batch of potent CpG ODN, including 10 B-class CpG ODNs or C-class CpG ODNs. The novel CpG ODN could be used as adjuvant for currently used vaccines and as monotherapeutics to treat cancer and infectious diseases.Among the novel CpG ODN we discovered, two of them, designated as YWODN 01 and YWODN 02 respectively, have been studied for their anti-tumor activities in mice inoculated with EMT6 breast cancer cells. We have found that YWODN 02, identified as a C-class CpG-ODN, displays more potent anti-tumor activity than YWODN01, identified as a B-class CpG-ODN. Inspiringly, after the tumor establishment, YWODN 02 injected through TDLN area induces dramatic retardation of tumor growth and complete tumor regression. This result suggests that YWODN 02, a C-class CpG ODN, may have the potential to be developed as a monotherapeutic medicament for the treatment of breast cancer through TDLN-injection.The data of CpG ODN in CVB-infected mice may indicated that when used for the treatment of viral-infected diseases in vivo, CpG ODN may function in a complicated way. These data indicated that its administration time, route and dosage, should be taken into full consideration.
引文
[1] Abe T, Hemmi H, Miyamoto H et al. Involvement of the toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus [J]. Journal of Virology 2005;79:2847-2858.
[2] Ahonen CL, Doxsee CL, McGurran SM et al. Combined TLR and CD40 triggering induces potent CD8(+) T cell expansion with variable dependence on type I IFN [J]. Journal of Experimental Medicine 2004;199:775-784.
[3] Amy M, Anthony J, Howard A, et al. Cytokines and acute pancreatitis [J]. Gastroenterology, 1996, 110 (2) : 639 6421
[4] American Cancer Society, Cancer Facts & Figures 2008 [R]. 250 Williams Street, NW, Atlanta, GA 30303-1002: American Cancer Society, 2008.
[5] Anderson BO. Breast conversation therapy (BCT) : surgery as the corner stone of multispecialty care [J]. Breast Cancer, 2000, 7
[6] Anichini A, Scarito A, Molla A et al. Differentiation of CD8(+) T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: Role of common gamma-chain cytokines [J]. Journal of Immunology 2003;171:2134-2141.
[7] Arthur DW, Morris MM, Vicini FA. Breast cancer: New radiation treatment options [J]. Oncology-New York 2004;18:1621-1629.
[8] Ayash-Rashkovsky M, Borkow G, Davis HL et al. Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection [J]. Faseb Journal 2005;19:1152-+.
[9] Ballas ZK, Krieg AM, Warren T et al. Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs [J]. Journal of Immunology. 2001;167:4878-4886.
[10] Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK Activity in Murine and Human Cells by CpG Motifs in Oligodeoxynucleotides and Bacterial DNA [J]. Journal of Immunology. 1996; 157: 1840-1845
[11] Barthelmes L, Al Awa A, Murali-Krishnan VP et al. The role of lymph node sampling and radiotherapy in the management of the axilla in early breast cancer [J]. Breast 2002;11:236-240.
[12] Bao MS, Zhang Y, Wan M et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide [J]. Clinical Immunology 2006;118:180-187.
[13] Bauer KR, Brown M, Cress RD et al. Descriptive analysis of estrogen receptor (ER)negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype-A population-based study from the California Cancer Registry [J]. Cancer 2007;109:1721-1728.
[14] Belkacemi Y, Comet B, Hannoun-Levi JM et al. Accelerated partial breast irradiation: a concept to individualize treatment in breast cancer [J]. Cancer Radiotherapie 2007;11:287-295.
[15] Becker Y. A point of view: HIV-1/AIDS is an allergy but CpG ODN treatments may inhibit virus replication and reactiate the adaptive immunity - Hypothesis and implications [J]. Virus Genes 2005;30:127-131.
[16] Bhatnagar AS. Review of the development of letrozole and its use in advanced breast cancer and in the neoadjuvant setting [J]. Breast 2006;15:S3-S13.
[17] Bird AP. CpG-rich islands and the function of DNA methylation [J]. Nature. 1986; 321(6067): 209–213
[18] Boér K, Láng I, Juhos E, et al., Adjuvant therapy of breast cancer with docetaxel-containing combination (TAC) [J]. Pathology oncology research, 2003;9(3):166-9
[19] Brunetti L, DeSantis ER. Treatment of viral myocarditis caused by coxsackievirus B. American journal of health-system pharmacy. 2008, 15;65(2):132-137
[20] Buhtoiarov IN, Lum HD, Berke G et al. Synergistic activation of macrophages via CD40 and TLR9 results in T cell independent antitumor effects [J]. Journal of Immunology 2006;176:309-318.
[21] Buzdar AU. 'Arimidex' (anastrozole) versus tamoxifen as adjuvant therapy in postmenopausal women with early breast cancer-efficacy overview [J]. Journal of Steroid Biochemistry and Molecular Biology 2003;86:399-403.
[22] Bumpers HL, Best IM, Norman D et al. Debilitating lymphedema of the upper extremity after treatment of breast cancer. American Journal of Clinical Oncology-Cancer Clinical Trials 2002;25:365-367.
[23] Bourquin C, Anz D, Zwiorek K et al. Targeting CpG oligonucleotides to the lymph node by nanoparticles elicits efficient antitumoral immunity [J]. Journalof Immunology 2008;181:2990-2998.
[24] Chanzy S, Mselati JC, Jourdain G et al. Group B coxsackie virus infection with fatal myocarditis in the neonate after early discharge from maternity unit [J]. Archives de Pediatrie 2005;12:618-619.
[25] Chui CS, Hong L, Hunt M et al. A simplified intensity modulated radiation therapy technique for the breast [J]. Medical Physics 2002;29:522-529.
[26] Carpentier A, Laigle-Donadey F, Zohar S et al. Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma [J]. Neuro-Oncology 2006;8:60-66.
[27] Carpentier AF, Xie J, Mokhtari K et al. Successful treatment of intracranial gliomas in rat by oligodeoxynucleotides containing CpG motifs [J]. Clinical Cancer Research 2000;6:2469-2473.
[28] Carpentier AF, Chen L, Maltonti F et al. Oligodeoxynucleotides containing CpG motifs can induce rejection of a neuroblastoma in mice [J]. Cancer Research 1999;59:5429-5432.
[29] Cao WP, Manicassamy S, Tang H et al. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3) K-mTOR-p70S6K pathway [J]. Nature Immunology 2008;9:1157-1164.
[30] Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity [J]. Nature Immunology 2004;5:1219-1226.
[31] Cong Z, Wan M, Wu XL et al. A CpG oligodeoxynucleotide inducing anti-coxsackie B3 virus activity in human peripheral blood mononuclear cells [J]. Fems Immunology and Medical Microbiology 2007;51:26-34.
[32] Cong YP, Song SS, Bhagat L et al. Self-stabilized CpG DNAs optimally activate human B cells and plasmacytoid dendritic cells [J]. Biochemical and Biophysical Research Communications 2003;310:1133-1139.
[33] de Alencar BCG, Araujo AFS, Penido MLO et al. Cross-priming of long lived protective CD8(+) T cells against Trypanosoma cruzi infection: Importance of a TLR9 agonist and CD4(+) T cells [J]. Vaccine 2007;25:6018-6027.
[34] Deml L, Schirmbeck R, Reimann J et al. Immunostimulatory CpG motifs trigger a T helper-1 immune response to human immunodeficiency virus type-1 (HIV-1) gp160 envelope proteins [J]. Clinical Chemistry and Laboratory Medicine 1999;37:199-204.
[35] Dumais N, Patrick A, Moss RB et al. Mucosal immunization with inactivated human immunodeficiency virus plus CpG oligodeoxynucleotides induces genital immune responses and protection against intravaginal challenge [J]. Journal of Infectious Diseases 2002;186:1098-1105.
[36] Friedberg JW, Kim H, McCauley M et al. Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha/beta-inducible gene expression, without significant toxicity [J]. Blood 2005;105:489-495.
[37] Friedberg JW, Kim H, McCauley M et al. Combination immunotherapy for non-Hodgkin's lymphoma (NHL) with a CpG oligonucleotide (1018 ISS) and rituximab: Biological responses through increased interferon-a/beta-inducible gene expression without significant toxicity [J]. Blood 2003;102:69A.
[38] Frisancho-Kiss S, Nyland JF, Davis SE et al. Sex differences in coxsackievirus B3-induced myocarditis: IL-12R beta 1 signaling and IFN-gamma increase inflammation in males independent from STAT4 [J]. Brain Research 2006;1126:139-147.
[39] Fattorossi A, Battaglia A, Ferrandina G et al. Lymphocyte composition of tumor draining lymph nodes from cervical and endometrial cancer patients [J]. Gynecologic Oncology 2004;92:106-115.
[40] Feldman AL, Arber DA, Pittaluga S et al. Clonally related follicular lymphomas and histiocytic/dendritic cell sarcomas: evidence for transdifferentiation of the follicular lymphoma clone [J]. Blood 2008;111:5433-5439.
[41] Fensterle J, Grode L, Hess J et al. Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun [J]. Journal of Immunology 1999;163:4510-4518.
[42] Finek J, Holubec L, Svoboda T et al. A Phase II Trial of Oral Vinorelbine and Capecitabine in Anthracycline Pretreated Patients with Metastatic Breast Cancer [J]. Anticancer Research 2009;29:667-670.
[43] Fujita N, Kagamu H, Yoshizawa H et al. CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells [J]. Journal of Immunology 2001;167:5678-5688.
[44] Gipponi M, Solari N, Di Somma FC et al. New fields of application of the sentinel lymph node biopsy in the pathologic staging of solid neoplasms: Reviewof literature and surgical perspectives [J]. Journal of Surgical Oncology 2004;85:171-179.
[45] Glue P, Fang JW, Rouzier-Panis R, et al. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data [J]. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther. 2000 Nov;68(5):556-67.
[46] Goss PE. Breast cancer prevention - clinical trials strategies involving aromatase inhibitors [J]. Journal of Steroid Biochemistry and Molecular Biology 2003;86:487-493
[47] Green J, Casabonne D, Newton R. Coxsackie B virus serology and Type 1 diabetes mellitus: a systematic review of published case-control studies [J]. Diabetic Medicine 2004;21:507-514.
[48] Gupta SK, Glue P, Jacobs S et al. Single-dose pharmacokinetics and tolerability of pegylated interferon-alpha 2b in young and elderly healthy subjects [J]. British Journal of Clinical Pharmacology 2003;56:131-134.
[49] Gursel M, Gursel I, Mostowski HS et al. CXCL16 influences the nature and specificity of CpG-Induced immune activation [J]. Journal of Immunology 2006;177:1575-1580.
[50] Hartmann E, Wollenberg B, Rothenfusser S et al. Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer [J]. Cancer Research 2003;63:6478-6487.
[51] Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA moth in human primary B cells [J]. Journal of Immunology 2000;164:944-952.
[52] Hargadon KM, Brinkman CC, Sheasley-O'Neill SL et al. Incomplete differentiation of antigen-specific CD8 T cells in tumor-draining lymph nodes [J]. Journal of Immunology 2006;177:6081-6090.
[53] Henderson IC, Berry DA, Demetri GD et al. Improved outcomes from adding sequential paclitaxel but not from escalating doxorubicin dose in an adjuvant chemotherapy regimen for patients with node-positive primary breast cancer [J]. Journal of Clinical Oncology 2003;21:976-983.
[54] Henke A, Zell R, Stelzner A. DNA vaccine-mediated immune responses in Coxsackie virus B3-infected mice [J]. Antiviral Research 2001;49:49-54.
[55] Heit A, Huster KM, Schmitz F et al. CpG-DNA aided cross-priming by cross-presenting B cells [J]. Journal of Immunology 2004;172:1501-1507.
[56] Heikenwalder M, Polymenidou M, Junt T et al. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration [J]. Nature Medicine 2004;10:187-192.
[57] Heckelsmiller, K., Rall, K., Beck, S., Schlamp, A., Seiderer, J., Jahrsdorfer, B., Krug, A., Rothenfusser, S., Endres, S., and Hartmann, G. Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model [J]. Journal of Immunology 169(7), 3892-3899. 10-1-2002.
[58] Heim A, Weiss S. Interferons in enteroviral heart disease: modulation of cytokine expression and antiviral activity [J]. Medical Microbiology and Immunology 2004;193:149-154.
[59] Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA [J]. Nature. 2000; 408(6813): 740-5
[60] Huber SA. T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes [J]. Cardiovascular Research 2000;45:579-587.
[61] Houssami N, Cuzick J, Dixon JM. The prevention, detection, and management of breast cancer [J]. Medical Journal of Australia 2006;184:230-234.
[62] Ito S, Ishii KI, Gursel M et al. CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection [J]. Journal of Immunology 2005;174:777-782.
[63] Isogawa M, Robek MD, Furuichi Y et al. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo [J]. Journal of Virology 2005;79:7269-7272.
[64] Ignatowicz-Pacyna A, Kornafel J. Advances in radiation therapy [J]. Advances in Clinical and Experimental Medicine 2007;16:695-700.
[65] Jiang W, Lederman MA, Mohner RJ et al. Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection [J]. Journal of Virology 2008;82:7837-7845.
[66] Jurk M, Vollmer J. Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation [J]. Biodrugs 2007;21:387-401.
[67] Jonasch E, Haluska FG. Interferon in oncological practice: Review of interferonbiology, clinical applications, and toxicities [J]. Oncologist 2001;6:34-55.
[68] Klinman DM, Currie D. Hierarchical recognition of CpG motifs expressed by immunostimulatory oligodeoxynucleotides [J]. Clinical and Experimental Immunology 2003;133:227-232.
[69] Kamstrup S, Verthelyi D, Klinman DM. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides [J]. Vet Microbiol. 2001; 78(4): 353-62
[70] Kandimalla ER, Bhagat L, Li YK et al. Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists [J]. Proceedings of the National Academy of Sciences of the United States of America 2005;102:6925-6930.
[71] Kandimalla ER, Bhagat L, Cong YP et al. Secondary structures in CpG oligonucleotides affect immunostimulatory activity [J]. Biochemical and Biophysical Research Communications 2003;306:948-953.
[72] Kandimalla ER, Bhagat L, Zhu FG et al. A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif [J]. Proceedings of the National Academy of Sciences of the United States of America 2003;100:14303-14308.
[73] Kandolf R, Sauter M, Aepinus C et al. Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system [J]. Virus Research 1999;62:149-158.
[74] Kawarada Y, Ganss R, Garbi N et al. NK- and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides [J]. Journal of Immunology 2001;167:5247-5253.
[75] Kerkmann M, Costa LT, Richter C et al. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells [J]. Journal of Biological Chemistry 2005;280:8086-8093.
[76] Kerkmann M, Rothenfusser S, Hornung V et al. Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type IIFN synthesis in human plasmacytoid dendritic cells [J]. Journal of Immunology 2003;170:4465-4474.
[77] Kilinc MO, Aulakh KS, Nair RE et al. Reversing tumor immune suppressionwith intratumoral IL-12: Activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8(+) T effectors [J]. Journal of Immunology 2006;177:6962-6973.
[78] Kilinc MO, Gu T, Harden JL et al. Central Role of Tumor-Associated CD8(+) T Effector/Memory Cells in Restoring Systemic Antitumor Immunity [J]. Journal of Immunology 2009;182:4217-4225.
[79] Kim JY, Jeon ES, Lim BK et al. Immunogenicity of a DNA vaccine for coxsackievirus B3 in mice: protective effects of capsid proteins against viral challenge [J]. Vaccine 2005;23:1672-1679.
[80] Kim R, Emi M, Tanabe K et al. Immunobiology of the sentinel lymph node and its potential role for antitumour immunity [J]. Lancet Oncology 2006;7:1006-1016.
[81] Kimura H, Iizasa T, Ishikawa A et al. Prospective phase II study of post-surgical adjuvant chemo-immunotherapy using autologous dendritic cells and activated killer cells from tissue culture of tumor-draining lymph nodes in primary lung cancer patients [J]. Anticancer Research 2008;28:1229-1238.
[82] Kimura H, Dobrenkov K, Iida T et al. Tumor-draining lymph nodes of primary lung cancer patients: A potent source of tumor-specific killer cells and dendritic cells [J]. Anticancer Research 2005;25:85-94.
[83] Kishimoto C, Crumpacker CS, Abelmann WH. Ribavirin treatment of murine coxsackievirus B3 myocarditis with analyses of lymphocyte subsets [J]. J Am Coll Cardiol. 1988 Nov;12(5):1334-41.
[84] Krieg AM. Therapeutic potential of Toll-like receptor 9 activation [J]. Nature Reviews Drug Discovery 2006;5:471-484.
[85] Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer [J]. Oncogene 2008;27:161-167.
[86] Krieg AM. Development of TLR9 agonists for cancer therapy [J]. Journal of Clinical Investigation 2007;117:1184-1194.
[87] Krug A, Rothenfusser S, Hornung V et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells [J]. European Journal of Immunology 2001;31:2154-2163.
[88] Krug A, Rothenfusser S, Selinger S et al. CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8T cells [J]. Journal of Immunology 2003;170:3468-3477.
[89] Kuchtey J, Chefalo PJ, Gray RC et al. Enhancement of dendritic cell antigen cross-presentation by CpG DNA involves type I IFN and stabilization of class I MHC mRNA [J]. Journal of Immunology 2005;175:2244-2251.
[90] Latz E, Schoenemeyer A, Visintin A et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome [J]. Nature Immunology 2004;5:190-198.
[91] Lake RA, Robinson BWS. Opinion - Immunotherapy and chemotherapy - a practical partnership [J]. Nature Reviews Cancer 2005;5:397-405.
[92] Lazovich D, Solomon CC, Thomas DB, et al. Breast conversation therapy in the United States from the1990National Institutes of Health Consen2susDevelopment Conference on the treatment of pa2 tientswith early stage invasive breast carcinoma [J]. Cancer, 1999, 86 (4) : 6282637.
[93] Le Bon A, Etchart N, Rossmann C et al. Cross-priming of CD8(+) T cells stimulated by virus-induced type I interferon [J]. Nature Immunology 2003;4:1009-1015.
[94] Leichman G, Gravenor D, Woytowitz D et al. CPG 7909, a TLR9 agonist, added to first line taxane/platinum for advanced non-small cell lung cancer, a randomized; controlled phase II study [J]. Journal of Clinical Oncology 2005;23:630S.
[95] Leifer CA, Verthelyi D, Klinman DM et al.. Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides [J]. J Immunother. 2003; 26(4): 313-9
[96] Li DP, Li H, Zhang PY et al. Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity [J]. European Journal of Immunology 2006;36:1324-1336.
[97] Li N, Fan XG, Chen ZH et al. Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B [J]. Apmis 2005;113:647-654.
[98] Liu YQ, Zhang XS, Zhang WD et al. Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8(+) cytotoxic T-cell activation and antitumor immunity [J]. Cancer Gene Therapy 2002;9:202-208.
[99] Liyanage UK, Moore TT, Joo HG et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients withpancreas or breast adenocarcinoma [J]. Journal of Immunology 2002;169:2756-2761.
[100] Lonsdorf AS, Kuekrek H, Stern BV et al. Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity [J]. Journal of Immunology 2003;171:3941-3946.
[101] Manegold C, Gravenor D, Woytowitz D et al. Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non-small-cell lung cancer [J]. Journal of Clinical Oncology 2008;26:3979-3986.
[102] Martinson JA, Tenorio AR, Montoya CJ et al. Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals [J]. Immunology 2007;120:526-535.
[103] Martino TA, Petric M, Weingartl H et al. The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus [J]. Virology 2000;271:99-108.
[104] Marshall JD, Fearon KL, Higgins D et al. Superior activity of the type C class of ISS in vitro and in vivo across multiple species [J]. Dna and Cell Biology 2005;24:63-72.
[105] Marshall JD, Fearon K, Abbate C et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions [J]. Journal of Leukocyte Biology 2003;73:781-792.
[106] Mamounas EP, Bryant J, Leinbersky B et al. Paclitaxel after doxorubicin plus cyclophosphamide as adjuvant chemotherapy for node-positive breast cancer: Results from NSABP B-28 [J]. Journal of Clinical Oncology 2005;23:3686-3696.
[107] Maurer T, Heit A, Hochrein H et al. CpG-DNA aided cross-presentation of soluble antigens by dendritic cells [J]. European Journal of Immunology 2002;32:2356-2364.
[108] Mastini C, Becker PD, Iezzi M et al. Intramammary application of non-methylated-CpG oligodeoxynucleotides (CpG) inhibits both local and systemic mammary carcinogenesis in female BALB/c Her-2/neu transgenic mice [J]. Current Cancer Drug Targets 2008;8:230-242.
[109] Mason JW, O’Connell JB, Herskowitz A et al. A clinical trial of immunosuppressive therapy for myocarditis [J]. New Engandl Journal of Medicine. 1995; 333:269-75.
[110] Mason KA, Neal R, Hunter N et al. CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors [J]. Radiotherapy and Oncology 2006;80:192-198.
[111] Mason KA, Ariga H, Neal R et al. Targeting Toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy [J]. Clinical Cancer Research 2005;11:361-369.
[112] McNamara DM, Holubkov R, Starling RC et al. Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy [J]. Circulation. 2001; 103:2254-9.
[113] Mellor AL, Baban B, Chandler PR et al. Cutting edge: CpG oligonucleotides induce splenic CD19(+) dendritic cells to acquire potent indoleamine 2,3-dioxygenase-dependent T cell regulatory functions via IFN type 1 signaling [J]. Journal of Immunology 2005;175:5601-5605.
[114] Meneses KD, Mcnees MP. Upper extremity lymphedema after treatment for breast cancer: A review of the literature [J]. Ostomy Wound Management 2007;53:16-29.
[115] Meng YX, Carpentier AF, Chen L et al. Successful combination of local CpG-ODN and radiotherapy in malignant glioma [J]. International Journal of Cancer 2005;116:992-997.
[116] Milas L, Mason KA, Ariga H et al. CpG oligodeoxynucleotide enhances tumor response to radiation [J]. Cancer Research 2004;64:5074-5077.
[117] Munn DH, Mellor AL. The tumor-draining lymph node as an immune-privileged site [J]. Immunological review 2006; Oct;213:146-58
[118] McHutchison JG, Bacon BR, Gordon SC et al. Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis c virus [J]. Hepatology 2007;46:1341-1349.
[119] Molenkamp BG, van Leeuwen PAM, van den Eertwegh AJM et al. Immunomodulation of the melanoma sentinel lymph node: A novel adjuvant therapeutic option [J]. Immunobiology 2006;211:651-661.
[120] Molenkamp BG, van Leeuwen PAM, Meijer S et al. Intradermal CpG-B activatesboth plasmacytoid and myeloid dendritic cells in the sentinel lymph node of melanoma patients [J]. Clinical Cancer Research 2007;13:2961-2969.
[121] Mortarini R, Piris A, Maurichi A et al. Lack of terminally differentiated tumor-specific CD8(+) T cells at tumor site in spite of antitumor immunity to self-antigens in human metastatic melanoma [J]. Cancer Research 2003;63:2535-2545.
[122] Morganti AG, Cilla S, Valentini V et al. Phase I-II studies on accelerated IMRT in breast carcinoma: Technical comparison and acute toxicity in 332 patients [J]. Radiotherapy and Oncology 2009;90:86-92.
[123] National Cancer Institute, SEER Stat Fact Sheets: cancer of the Breast [R]. Bethesda: NCI, 2008
[124] Nathanson SD. Insights into the mechanisms of lymph node metastasis [J]. Cancer 2003;98:413-423.
[125] Nakano H, Yanagita M, Gunn MD. CD11c(+)B220(+)Gr-1(+) cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells [J]. Journal of Experimental Medicine 2001;194:1171-1178.
[126] Nagorsen D, Voigt S, Berg E et al. Tumor-infiltrating macrophages and dendritic cells in human colorectal cancer: relation to local regulatory T cells, systemic T-cell response against tumor-associated antigens and survival [J]. Journal of Translational Medicine 2007;5.
[127] Nichani AK, Kaushik RS, Mena A et al. CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep [J]. Cell Immunol. 2004; 227(1): 24-37
[128] Ning L, Fan XG, Chen ZH et al. Inhibition of the hepatitis B virus replication in vitro by an oligodeoxynucleotide containing cytidine-guano sine motifs [J]. Immunology Letters 2006;102:60-66.
[129] Nowroozalizadeh S, Jansson M, Adamsson J et al. Suppression of HIV replication in vitro by CpG and CpG conjugated to the non toxic B subunit of cholera toxin [J]. Current Hiv Research 2008;6:230-238.
[130] Olbrich ARM, Schimmer S, Heeg K et al. Effective postexposure treatment of retrovirus-induced disease with immunostimulatory DNA containing CpG motifs [J]. Journal of Virology 2002;76:11397-11404.
[131] Olbrich ARM, Schimmer S, Dittmer U. Preinfection treatment of resistant mice with CpG oligodeoxynucleotides renders them susceptible to friend retrovirus-induced leukemia [J]. Journal of Virology 2003;77:10658-10662.
[132] Opavsky A, Penninger J, Aitken K et al. Host susceptibility to CVB3 myocarditis in T cell knockout mice is associated with distinct myocardial cytokine expression repertoire [J]. Circulation 1999;100:429.
[133] Padalko E, Nuyens D, De Palma A et al. The interferon inducer ampligen [poly(I)-poly(C12U)] markedly protects mice against coxsackie 133 virus-induced myocarditis [J]. Antimicrobial Agents and Chemotherapy 2004;48:267-274.
[134] Pashenkov M, Goess G, Wagner C et al. Phase II trial of a Toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma [J]. Journal of Clinical Oncology 2006;24:5716-5724.
[135] Pasare C, Medzhitov R. Toll-like receptors: Linking innate and adaptive immunity. Mechanisms of Lymphocyte Activation and Immune Regulation X [J]: Innate Immunity 2005;560:11-18.
[136] Peng WM, Yu CF, Allam JP et al. Inhibitory oligodeoxynucleotides downregulate herpes simplex virus-induced plasmacytoid dendritic cell type I interferon production and modulate cell function [J]. Human Immunology 2007;68:879-887.
[137] Piccart M, Hamilton A. HER2/neu as a predictive factor in breast cancer [J]. European Journal of Cancer 1999;35:S84.
[138] Poindexter NJ, Sahin A, Hunt KK et al. Analysis of dendritic cells in tumor-free and tumor-containing sentinel lymph nodes from patients with breast cancer [J]. Breast Cancer Research 2004;6:R408-R415.
[139] Rayburn ER, Wang W, Zhang Z et al. Experimental therapy of prostate cancer with an immunomodulatory oligonucleotide: Effects on tumor growth, apoptosis, proliferation, and potentiation of chemotherapy [J]. Prostate 2006;66:1653-1663.
[140] Rayburn ER, Wang W, Zhang RW et al. Experimental therapy for colon cancer: Anti-cancer effects of TLR9 agonism, combination with other therapeutic modalities, and dependence upon p53 [J]. International Journal of Oncology 2007;30:1511-1519.
[141] Robinson NMK, Zhang HY, Bevan AL et al. Induction of myocardial nitric oxide synthase by Coxsackie B3 virus in mice [J]. European Journal of Clinical Investigation 1999;29:700-707.
[142] Rothenfusser S, Hornung V, Ayyoub M et al. CpG-A and CpG-Boligonucleotides differentially enhance human peptide-specific primary and memory CD8(+) T-cell responses in vitro [J]. Blood 2004;103:2162-2169.
[143] Rosset MB, Ballerini C, Gregoire S et al. Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice [J]. Journal of Immunology 2004;172:5168-5174.
[144] Sfondrini L, Besusso D, Rumio C et al. Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA [J]. Faseb Journal 2002;16:1749-1754.
[145] Shu S, Cochran AJ, Huang RR et al. Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy [J]. Cancer and Metastasis Reviews 2006;25:233-242.
[146] Speiser DE, Lienard D, Rufer N et al. Rapid and strong human CD8(+) T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909 [J]. Journal of Clinical Investigation 2005;115:739-746.
[147] Srinivasan S, Parsa V, Liu CY et al. Trastuzumab-induced hepatotoxicity [J]. Annals of Pharmacotherapy 2008;42:1497-1501.
[148] Sanders ME, Scroggins T, Ampil FL et al. Accelerated partial breast irradiation in early-stage breast cancer [J]. Journal of Clinical Oncology 2007;25:996-1002.
[149] Santin AD. Lymph node metastases - The importance of the microenvironment [J]. Cancer 2000;88:175-179.
[150] Saloustros E, Mavroudis D, Georgoulias V. Paclitaxel and docetaxel in the treatment of breast cancer [J]. Expert Opinion on Pharmacotherapy 2008;9:2603-2616.
[151] Sasse A, Wallich M, Ding Z et al. Coxsackie-and-adenovirus receptor mRNA expression in human heart failure [J]. Journal of Gene Medicine 2003;5:876-882.
[152] Sethi S, Lipford G, Wagner H et al. Postexposure prophylaxis against prion disease with a stimulator of innate immunity [J]. Lancet 2002;360:229-230.
[153] Sivori S, Carlomagno S, Moretta L et al. Comparison of different CpG oligodeoxynucleotide classes for their capability to stimulate human NK cells [J]. European Journal of Immunology 2006;36:961-967.
[154] Sun SQ, Zhang XH, Tough DF et al. Type I interferon-mediated stimulation of T cells by CgG DNA [J]. Journal of Experimental Medicine 1998;188:2335-2342.
[155] Schlaepfer E, Audige A, von Beust B et al. CpG oligodeoxynucleotides blockhuman immunodeficiency virus type 1 replication in human lymphoid tissue infected ex vivo [J]. Journal of Virology 2004;78:12344-12354.
[156] Scheller C, Ullrich A, McPherson K et al. CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells [J]. Journal of Biological Chemistry 2004;279:21897-21902.
[157] Schwaab T, Weiss JE, Schned AR et al. Dendritic cell infiltration in colon cancer [J]. Journal of Immunotherapy 2001;24:130-137.
[158] Scott LJ, Keam SJ. Letrozole: In postmenopausal hormone-responsive early-stage breast cancer [J]. Drugs 2006;66:353-362.
[159] Sohn WJ, Lee KW, Choi SY et al. CpG-oligodeoxynucleotide protects immune cells from gamma-irradiation-induced cell death [J]. Molecular Immunology 2006;43:1163-1171.
[160] Thompson JA, Kuzel T, Bukowski R et al. Phase lb trial of a targeted TLR9 CpG immunomodulator (CPG 7909) in advanced renal cell carcinoma (RCC) [J]. Journal of Clinical Oncology 2004;22:417S.
[161] Takahashi K, Nishikawa Y, Sato H et al. Dendritic cells interacting mainly with B cells in the lymphoepithelial symbiosis of the human palatine tonsil [J]. Virchows Archiv 2006;448:623-629.
[162] Takahashi K, Kenji A, Norihiro T et al. Morphological interactions of interdigitating dendritic cells with B and T cells in human mesenteric lymph nodes [J]. American Journal of Pathology 2001;159:131-138.
[163] Takeshita F, Gursel I, Ishii KJ et al. Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9 [J]. Seminars in Immunology 2004;16:17-22.
[164] Teleshova N, Kenney J, Jones J et al. CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting Simian immunodeficiency virus-specific T cells [J]. Journal of Immunology 2004;173:1647-1657.
[165] Teleshova N, Kenney J, Van Nest G et al. Local and systemic effects of intranodally injected CpG-C immunostimulatory-oligodeoxyribonucleotides in macaques [J]. Journal of Immunology 2006;177:8531-8541.
[166] Tsuge T, Yamakawa M, Tsukamoto M. Infiltrating dendritic/Langerhans cells inprimary breast cancer [J]. Breast Cancer Research and Treatment 2000;59:141-152.
[167] Ugolini C, Basolo F, Proietti A et al. Lymphocyte and immature dendritic cell infiltrates in differentiated, poorly differentiated, and undifferentiated thyroid carcinoma [J]. Thyroid 2007;17:389-393.
[168] Van der Stede Y, Verdonck F, Vancaeneghem S et al. CpG-oligodinucleotides as an effective adjuvant in pigs for intramuscular immunizations [J]. Veterinary Immunology and Immunopathology 2002;86:31-41.
[169] van der Most RG, Currie A, Robinson BWS et al. Cranking the immunologic engine with chemotherapy: Using context to drive tumor antigen cross-presentation towards useful antitumor immunity [J]. Cancer Research 2006;66:601-604.
[170] van Ojik HH, Bevaart L, Dahle CE et al. CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations [J]. Cancer Research 2003;63:5595-5600.
[171] Verthelyi D, Ishii KJ, Gursel M et al. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs [J]. Journal of Immunology 2001;166:2372-2377.
[172] Verthelyi D, Gursel M, Kenney RT et al. CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection [J]. Journal of Immunology 2003;170:4717-4723.
[173] Vincent IE, Lucifora J, Durantel D et al. Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro [J]. Antiviral Therapy 2009;14:131-135.
[174] Viale PH, Yamamoto DS. Cardiovascular toxicity associated with cancer treatment [J]. Clinical Journal of Oncology Nursing 2008;12:627-638.
[175] Vuylsteke RJCL, Molenkamp BG, Gietema HA et al. Local administration of granulocyte/macrophage colony-stimulating factor increases the number and activation state of dendritic cells in the sentinel lymph node of early-stage melanoma [J]. Cancer Research 2004;64:8456-8460.
[176] Vuylsteke RJCL, Molenkamp BG, van Leeuwen PAM et al. Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF-treated stage I melanoma patients is associated with high myeloid dendritic cell content [J].Clinical Cancer Research 2006;12:2826-2833.
[177] Vollmer J, Weeratna R, Payette P et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities [J]. European Journal of Immunology 2004;34:251-262.
[178] Wang H, Rayburn ER, Wang W et al. Immunomodulatory oligonucleotides as novel therapy for breast cancer: pharmacokinetics, in vitro and in vivo anticancer activity, and potentiation of antibody therapy [J]. Molecular Cancer Therapeutics 2006;5:2106-2114.
[179] Wang XJ, Bao MS, Wan M et al. A CpG oligodeoxynucleotide acts as a potent adjuvant for inactivated rabies virus vaccine [J]. Vaccine 2008;26:1893-1901.
[180] Wang XS, Sheng Z, Ruan YB,et al. CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma [J]. World J Gastroenterol. 2005, 11(8):1220-1224
[181] Wang YX, Da Cunha V, Vincelette J et al. Antiviral and myocyte protective effects of murine interferon-beta and -alpha 2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice [J]. American Journal of Physiology-Heart and Circulatory Physiology 2007;293:H69-H76
[182] Weeratna RD, Bourne LL, Sullivan SM et al. Combination of a new TLR9 agonist immunomodulator (CpG 7909) and paclitaxel for treatment of metastatic Lewis Lung Carcinoma (LLC) [J]. Journal of Clinical Oncology 2004;22:702S.
[183] Wernette CM, Smith BF, Barksdale ZL et al. CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation [J]. Vet Immunol Immunopathol. 2002; 84(3-4): 223-36
[184] Wingender G, Garbi N, Schumak B et al. Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO [J]. European Journal of Immunology 2006;36:12-20.
[185] Xiong ZM, Gharagozlou S, Vengco I et al. Effective CpG immunotherapy of breast carcinoma prevents but fails to eradicate established brain metastasis [J]. Clinical Cancer Research 2008;14:5484-5493.
[186] Yamamoto S, Yamamoto T, Kataoka T, et al. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity [J]. Journal ofImmunology 1992; 148(12):4072-6
[187] Yasuda K, Rutz M, Schlatter B et al. CpG motif-independent activation of TLR9 upon endosomal translocation of "natural" phosphodiester DNA [J]. European Journal of Immunology 2006;36:431-436.
[188] Yasuda K, Yu P, Kirschning CJ et al. Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways [J]. Journal of Immunology 2005;174:6129-6136.
[189] Yoshizawa H, Chang AE, Shu SY. Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells [J].Cancer Res. 1992 Mar 1;52(5):1129-36
[190] Yoshizawa H, Chang AE, Shu SY. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2 [J]. J Immunol. 1991 Jul 15;147(2):729-37
[191] Yu D, Putta MR, Bhagat L et al. Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction [J]. Antimicrobial Agents and Chemotherapy 2008;52:4320-4325.
[192] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction [J]. Nucleic Acids Research 2003;31:3406-3415.
[193]陈忠明,田少敏,阮康成。高压力制备柯萨奇病毒疫苗[J]。生物化学与生物物理学报,2001,33(1):128～130
[194]李锋,张立红,田淑芝。干扰素治疗小儿病毒性心肌炎的临床和免疫学观察[J]。医学综述,2008,14(12):1915-1917。
[195]刘文春,黄星原。黄芪注射液对病毒性心肌炎小鼠生存率及预后的影响[J],湖北中医杂志,2004,26(7):10-11
[196]王雪峰,郭津津,魏克伦等。小柴胡汤分解剂抗柯萨奇B3病毒感染及其对心肌炎防治作用的研究[J],中国医科大学学报,2000,29(2):115-117
[197]徐海燕,马沛然。西洋参对小鼠病毒性心肌炎的疗效及机制[J],山东中医药大学学报,2002,26(6):458-461
[2] Ahonen CL, Doxsee CL, McGurran SM et al. Combined TLR and CD40 triggering induces potent CD8(+) T cell expansion with variable dependence on type I IFN [J]. Journal of Experimental Medicine 2004;199:775-784.
[3] Amy M, Anthony J, Howard A, et al. Cytokines and acute pancreatitis [J]. Gastroenterology, 1996, 110 (2) : 639 6421
[4] American Cancer Society, Cancer Facts & Figures 2008 [R]. 250 Williams Street, NW, Atlanta, GA 30303-1002: American Cancer Society, 2008.
[5] Anderson BO. Breast conversation therapy (BCT) : surgery as the corner stone of multispecialty care [J]. Breast Cancer, 2000, 7
[6] Anichini A, Scarito A, Molla A et al. Differentiation of CD8(+) T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: Role of common gamma-chain cytokines [J]. Journal of Immunology 2003;171:2134-2141.
[7] Arthur DW, Morris MM, Vicini FA. Breast cancer: New radiation treatment options [J]. Oncology-New York 2004;18:1621-1629.
[8] Ayash-Rashkovsky M, Borkow G, Davis HL et al. Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection [J]. Faseb Journal 2005;19:1152-+.
[9] Ballas ZK, Krieg AM, Warren T et al. Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs [J]. Journal of Immunology. 2001;167:4878-4886.
[10] Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK Activity in Murine and Human Cells by CpG Motifs in Oligodeoxynucleotides and Bacterial DNA [J]. Journal of Immunology. 1996; 157: 1840-1845
[11] Barthelmes L, Al Awa A, Murali-Krishnan VP et al. The role of lymph node sampling and radiotherapy in the management of the axilla in early breast cancer [J]. Breast 2002;11:236-240.
[12] Bao MS, Zhang Y, Wan M et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide [J]. Clinical Immunology 2006;118:180-187.
[13] Bauer KR, Brown M, Cress RD et al. Descriptive analysis of estrogen receptor (ER)negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype-A population-based study from the California Cancer Registry [J]. Cancer 2007;109:1721-1728.
[14] Belkacemi Y, Comet B, Hannoun-Levi JM et al. Accelerated partial breast irradiation: a concept to individualize treatment in breast cancer [J]. Cancer Radiotherapie 2007;11:287-295.
[15] Becker Y. A point of view: HIV-1/AIDS is an allergy but CpG ODN treatments may inhibit virus replication and reactiate the adaptive immunity - Hypothesis and implications [J]. Virus Genes 2005;30:127-131.
[16] Bhatnagar AS. Review of the development of letrozole and its use in advanced breast cancer and in the neoadjuvant setting [J]. Breast 2006;15:S3-S13.
[17] Bird AP. CpG-rich islands and the function of DNA methylation [J]. Nature. 1986; 321(6067): 209–213
[18] Boér K, Láng I, Juhos E, et al., Adjuvant therapy of breast cancer with docetaxel-containing combination (TAC) [J]. Pathology oncology research, 2003;9(3):166-9
[19] Brunetti L, DeSantis ER. Treatment of viral myocarditis caused by coxsackievirus B. American journal of health-system pharmacy. 2008, 15;65(2):132-137
[20] Buhtoiarov IN, Lum HD, Berke G et al. Synergistic activation of macrophages via CD40 and TLR9 results in T cell independent antitumor effects [J]. Journal of Immunology 2006;176:309-318.
[21] Buzdar AU. 'Arimidex' (anastrozole) versus tamoxifen as adjuvant therapy in postmenopausal women with early breast cancer-efficacy overview [J]. Journal of Steroid Biochemistry and Molecular Biology 2003;86:399-403.
[22] Bumpers HL, Best IM, Norman D et al. Debilitating lymphedema of the upper extremity after treatment of breast cancer. American Journal of Clinical Oncology-Cancer Clinical Trials 2002;25:365-367.
[23] Bourquin C, Anz D, Zwiorek K et al. Targeting CpG oligonucleotides to the lymph node by nanoparticles elicits efficient antitumoral immunity [J]. Journalof Immunology 2008;181:2990-2998.
[24] Chanzy S, Mselati JC, Jourdain G et al. Group B coxsackie virus infection with fatal myocarditis in the neonate after early discharge from maternity unit [J]. Archives de Pediatrie 2005;12:618-619.
[25] Chui CS, Hong L, Hunt M et al. A simplified intensity modulated radiation therapy technique for the breast [J]. Medical Physics 2002;29:522-529.
[26] Carpentier A, Laigle-Donadey F, Zohar S et al. Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma [J]. Neuro-Oncology 2006;8:60-66.
[27] Carpentier AF, Xie J, Mokhtari K et al. Successful treatment of intracranial gliomas in rat by oligodeoxynucleotides containing CpG motifs [J]. Clinical Cancer Research 2000;6:2469-2473.
[28] Carpentier AF, Chen L, Maltonti F et al. Oligodeoxynucleotides containing CpG motifs can induce rejection of a neuroblastoma in mice [J]. Cancer Research 1999;59:5429-5432.
[29] Cao WP, Manicassamy S, Tang H et al. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3) K-mTOR-p70S6K pathway [J]. Nature Immunology 2008;9:1157-1164.
[30] Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity [J]. Nature Immunology 2004;5:1219-1226.
[31] Cong Z, Wan M, Wu XL et al. A CpG oligodeoxynucleotide inducing anti-coxsackie B3 virus activity in human peripheral blood mononuclear cells [J]. Fems Immunology and Medical Microbiology 2007;51:26-34.
[32] Cong YP, Song SS, Bhagat L et al. Self-stabilized CpG DNAs optimally activate human B cells and plasmacytoid dendritic cells [J]. Biochemical and Biophysical Research Communications 2003;310:1133-1139.
[33] de Alencar BCG, Araujo AFS, Penido MLO et al. Cross-priming of long lived protective CD8(+) T cells against Trypanosoma cruzi infection: Importance of a TLR9 agonist and CD4(+) T cells [J]. Vaccine 2007;25:6018-6027.
[34] Deml L, Schirmbeck R, Reimann J et al. Immunostimulatory CpG motifs trigger a T helper-1 immune response to human immunodeficiency virus type-1 (HIV-1) gp160 envelope proteins [J]. Clinical Chemistry and Laboratory Medicine 1999;37:199-204.
[35] Dumais N, Patrick A, Moss RB et al. Mucosal immunization with inactivated human immunodeficiency virus plus CpG oligodeoxynucleotides induces genital immune responses and protection against intravaginal challenge [J]. Journal of Infectious Diseases 2002;186:1098-1105.
[36] Friedberg JW, Kim H, McCauley M et al. Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha/beta-inducible gene expression, without significant toxicity [J]. Blood 2005;105:489-495.
[37] Friedberg JW, Kim H, McCauley M et al. Combination immunotherapy for non-Hodgkin's lymphoma (NHL) with a CpG oligonucleotide (1018 ISS) and rituximab: Biological responses through increased interferon-a/beta-inducible gene expression without significant toxicity [J]. Blood 2003;102:69A.
[38] Frisancho-Kiss S, Nyland JF, Davis SE et al. Sex differences in coxsackievirus B3-induced myocarditis: IL-12R beta 1 signaling and IFN-gamma increase inflammation in males independent from STAT4 [J]. Brain Research 2006;1126:139-147.
[39] Fattorossi A, Battaglia A, Ferrandina G et al. Lymphocyte composition of tumor draining lymph nodes from cervical and endometrial cancer patients [J]. Gynecologic Oncology 2004;92:106-115.
[40] Feldman AL, Arber DA, Pittaluga S et al. Clonally related follicular lymphomas and histiocytic/dendritic cell sarcomas: evidence for transdifferentiation of the follicular lymphoma clone [J]. Blood 2008;111:5433-5439.
[41] Fensterle J, Grode L, Hess J et al. Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun [J]. Journal of Immunology 1999;163:4510-4518.
[42] Finek J, Holubec L, Svoboda T et al. A Phase II Trial of Oral Vinorelbine and Capecitabine in Anthracycline Pretreated Patients with Metastatic Breast Cancer [J]. Anticancer Research 2009;29:667-670.
[43] Fujita N, Kagamu H, Yoshizawa H et al. CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells [J]. Journal of Immunology 2001;167:5678-5688.
[44] Gipponi M, Solari N, Di Somma FC et al. New fields of application of the sentinel lymph node biopsy in the pathologic staging of solid neoplasms: Reviewof literature and surgical perspectives [J]. Journal of Surgical Oncology 2004;85:171-179.
[45] Glue P, Fang JW, Rouzier-Panis R, et al. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data [J]. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther. 2000 Nov;68(5):556-67.
[46] Goss PE. Breast cancer prevention - clinical trials strategies involving aromatase inhibitors [J]. Journal of Steroid Biochemistry and Molecular Biology 2003;86:487-493
[47] Green J, Casabonne D, Newton R. Coxsackie B virus serology and Type 1 diabetes mellitus: a systematic review of published case-control studies [J]. Diabetic Medicine 2004;21:507-514.
[48] Gupta SK, Glue P, Jacobs S et al. Single-dose pharmacokinetics and tolerability of pegylated interferon-alpha 2b in young and elderly healthy subjects [J]. British Journal of Clinical Pharmacology 2003;56:131-134.
[49] Gursel M, Gursel I, Mostowski HS et al. CXCL16 influences the nature and specificity of CpG-Induced immune activation [J]. Journal of Immunology 2006;177:1575-1580.
[50] Hartmann E, Wollenberg B, Rothenfusser S et al. Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer [J]. Cancer Research 2003;63:6478-6487.
[51] Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA moth in human primary B cells [J]. Journal of Immunology 2000;164:944-952.
[52] Hargadon KM, Brinkman CC, Sheasley-O'Neill SL et al. Incomplete differentiation of antigen-specific CD8 T cells in tumor-draining lymph nodes [J]. Journal of Immunology 2006;177:6081-6090.
[53] Henderson IC, Berry DA, Demetri GD et al. Improved outcomes from adding sequential paclitaxel but not from escalating doxorubicin dose in an adjuvant chemotherapy regimen for patients with node-positive primary breast cancer [J]. Journal of Clinical Oncology 2003;21:976-983.
[54] Henke A, Zell R, Stelzner A. DNA vaccine-mediated immune responses in Coxsackie virus B3-infected mice [J]. Antiviral Research 2001;49:49-54.
[55] Heit A, Huster KM, Schmitz F et al. CpG-DNA aided cross-priming by cross-presenting B cells [J]. Journal of Immunology 2004;172:1501-1507.
[56] Heikenwalder M, Polymenidou M, Junt T et al. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration [J]. Nature Medicine 2004;10:187-192.
[57] Heckelsmiller, K., Rall, K., Beck, S., Schlamp, A., Seiderer, J., Jahrsdorfer, B., Krug, A., Rothenfusser, S., Endres, S., and Hartmann, G. Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model [J]. Journal of Immunology 169(7), 3892-3899. 10-1-2002.
[58] Heim A, Weiss S. Interferons in enteroviral heart disease: modulation of cytokine expression and antiviral activity [J]. Medical Microbiology and Immunology 2004;193:149-154.
[59] Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA [J]. Nature. 2000; 408(6813): 740-5
[60] Huber SA. T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes [J]. Cardiovascular Research 2000;45:579-587.
[61] Houssami N, Cuzick J, Dixon JM. The prevention, detection, and management of breast cancer [J]. Medical Journal of Australia 2006;184:230-234.
[62] Ito S, Ishii KI, Gursel M et al. CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection [J]. Journal of Immunology 2005;174:777-782.
[63] Isogawa M, Robek MD, Furuichi Y et al. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo [J]. Journal of Virology 2005;79:7269-7272.
[64] Ignatowicz-Pacyna A, Kornafel J. Advances in radiation therapy [J]. Advances in Clinical and Experimental Medicine 2007;16:695-700.
[65] Jiang W, Lederman MA, Mohner RJ et al. Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection [J]. Journal of Virology 2008;82:7837-7845.
[66] Jurk M, Vollmer J. Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation [J]. Biodrugs 2007;21:387-401.
[67] Jonasch E, Haluska FG. Interferon in oncological practice: Review of interferonbiology, clinical applications, and toxicities [J]. Oncologist 2001;6:34-55.
[68] Klinman DM, Currie D. Hierarchical recognition of CpG motifs expressed by immunostimulatory oligodeoxynucleotides [J]. Clinical and Experimental Immunology 2003;133:227-232.
[69] Kamstrup S, Verthelyi D, Klinman DM. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides [J]. Vet Microbiol. 2001; 78(4): 353-62
[70] Kandimalla ER, Bhagat L, Li YK et al. Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists [J]. Proceedings of the National Academy of Sciences of the United States of America 2005;102:6925-6930.
[71] Kandimalla ER, Bhagat L, Cong YP et al. Secondary structures in CpG oligonucleotides affect immunostimulatory activity [J]. Biochemical and Biophysical Research Communications 2003;306:948-953.
[72] Kandimalla ER, Bhagat L, Zhu FG et al. A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif [J]. Proceedings of the National Academy of Sciences of the United States of America 2003;100:14303-14308.
[73] Kandolf R, Sauter M, Aepinus C et al. Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system [J]. Virus Research 1999;62:149-158.
[74] Kawarada Y, Ganss R, Garbi N et al. NK- and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides [J]. Journal of Immunology 2001;167:5247-5253.
[75] Kerkmann M, Costa LT, Richter C et al. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells [J]. Journal of Biological Chemistry 2005;280:8086-8093.
[76] Kerkmann M, Rothenfusser S, Hornung V et al. Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type IIFN synthesis in human plasmacytoid dendritic cells [J]. Journal of Immunology 2003;170:4465-4474.
[77] Kilinc MO, Aulakh KS, Nair RE et al. Reversing tumor immune suppressionwith intratumoral IL-12: Activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8(+) T effectors [J]. Journal of Immunology 2006;177:6962-6973.
[78] Kilinc MO, Gu T, Harden JL et al. Central Role of Tumor-Associated CD8(+) T Effector/Memory Cells in Restoring Systemic Antitumor Immunity [J]. Journal of Immunology 2009;182:4217-4225.
[79] Kim JY, Jeon ES, Lim BK et al. Immunogenicity of a DNA vaccine for coxsackievirus B3 in mice: protective effects of capsid proteins against viral challenge [J]. Vaccine 2005;23:1672-1679.
[80] Kim R, Emi M, Tanabe K et al. Immunobiology of the sentinel lymph node and its potential role for antitumour immunity [J]. Lancet Oncology 2006;7:1006-1016.
[81] Kimura H, Iizasa T, Ishikawa A et al. Prospective phase II study of post-surgical adjuvant chemo-immunotherapy using autologous dendritic cells and activated killer cells from tissue culture of tumor-draining lymph nodes in primary lung cancer patients [J]. Anticancer Research 2008;28:1229-1238.
[82] Kimura H, Dobrenkov K, Iida T et al. Tumor-draining lymph nodes of primary lung cancer patients: A potent source of tumor-specific killer cells and dendritic cells [J]. Anticancer Research 2005;25:85-94.
[83] Kishimoto C, Crumpacker CS, Abelmann WH. Ribavirin treatment of murine coxsackievirus B3 myocarditis with analyses of lymphocyte subsets [J]. J Am Coll Cardiol. 1988 Nov;12(5):1334-41.
[84] Krieg AM. Therapeutic potential of Toll-like receptor 9 activation [J]. Nature Reviews Drug Discovery 2006;5:471-484.
[85] Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer [J]. Oncogene 2008;27:161-167.
[86] Krieg AM. Development of TLR9 agonists for cancer therapy [J]. Journal of Clinical Investigation 2007;117:1184-1194.
[87] Krug A, Rothenfusser S, Hornung V et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells [J]. European Journal of Immunology 2001;31:2154-2163.
[88] Krug A, Rothenfusser S, Selinger S et al. CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8T cells [J]. Journal of Immunology 2003;170:3468-3477.
[89] Kuchtey J, Chefalo PJ, Gray RC et al. Enhancement of dendritic cell antigen cross-presentation by CpG DNA involves type I IFN and stabilization of class I MHC mRNA [J]. Journal of Immunology 2005;175:2244-2251.
[90] Latz E, Schoenemeyer A, Visintin A et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome [J]. Nature Immunology 2004;5:190-198.
[91] Lake RA, Robinson BWS. Opinion - Immunotherapy and chemotherapy - a practical partnership [J]. Nature Reviews Cancer 2005;5:397-405.
[92] Lazovich D, Solomon CC, Thomas DB, et al. Breast conversation therapy in the United States from the1990National Institutes of Health Consen2susDevelopment Conference on the treatment of pa2 tientswith early stage invasive breast carcinoma [J]. Cancer, 1999, 86 (4) : 6282637.
[93] Le Bon A, Etchart N, Rossmann C et al. Cross-priming of CD8(+) T cells stimulated by virus-induced type I interferon [J]. Nature Immunology 2003;4:1009-1015.
[94] Leichman G, Gravenor D, Woytowitz D et al. CPG 7909, a TLR9 agonist, added to first line taxane/platinum for advanced non-small cell lung cancer, a randomized; controlled phase II study [J]. Journal of Clinical Oncology 2005;23:630S.
[95] Leifer CA, Verthelyi D, Klinman DM et al.. Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides [J]. J Immunother. 2003; 26(4): 313-9
[96] Li DP, Li H, Zhang PY et al. Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity [J]. European Journal of Immunology 2006;36:1324-1336.
[97] Li N, Fan XG, Chen ZH et al. Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B [J]. Apmis 2005;113:647-654.
[98] Liu YQ, Zhang XS, Zhang WD et al. Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8(+) cytotoxic T-cell activation and antitumor immunity [J]. Cancer Gene Therapy 2002;9:202-208.
[99] Liyanage UK, Moore TT, Joo HG et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients withpancreas or breast adenocarcinoma [J]. Journal of Immunology 2002;169:2756-2761.
[100] Lonsdorf AS, Kuekrek H, Stern BV et al. Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity [J]. Journal of Immunology 2003;171:3941-3946.
[101] Manegold C, Gravenor D, Woytowitz D et al. Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non-small-cell lung cancer [J]. Journal of Clinical Oncology 2008;26:3979-3986.
[102] Martinson JA, Tenorio AR, Montoya CJ et al. Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals [J]. Immunology 2007;120:526-535.
[103] Martino TA, Petric M, Weingartl H et al. The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus [J]. Virology 2000;271:99-108.
[104] Marshall JD, Fearon KL, Higgins D et al. Superior activity of the type C class of ISS in vitro and in vivo across multiple species [J]. Dna and Cell Biology 2005;24:63-72.
[105] Marshall JD, Fearon K, Abbate C et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions [J]. Journal of Leukocyte Biology 2003;73:781-792.
[106] Mamounas EP, Bryant J, Leinbersky B et al. Paclitaxel after doxorubicin plus cyclophosphamide as adjuvant chemotherapy for node-positive breast cancer: Results from NSABP B-28 [J]. Journal of Clinical Oncology 2005;23:3686-3696.
[107] Maurer T, Heit A, Hochrein H et al. CpG-DNA aided cross-presentation of soluble antigens by dendritic cells [J]. European Journal of Immunology 2002;32:2356-2364.
[108] Mastini C, Becker PD, Iezzi M et al. Intramammary application of non-methylated-CpG oligodeoxynucleotides (CpG) inhibits both local and systemic mammary carcinogenesis in female BALB/c Her-2/neu transgenic mice [J]. Current Cancer Drug Targets 2008;8:230-242.
[109] Mason JW, O’Connell JB, Herskowitz A et al. A clinical trial of immunosuppressive therapy for myocarditis [J]. New Engandl Journal of Medicine. 1995; 333:269-75.
[110] Mason KA, Neal R, Hunter N et al. CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors [J]. Radiotherapy and Oncology 2006;80:192-198.
[111] Mason KA, Ariga H, Neal R et al. Targeting Toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy [J]. Clinical Cancer Research 2005;11:361-369.
[112] McNamara DM, Holubkov R, Starling RC et al. Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy [J]. Circulation. 2001; 103:2254-9.
[113] Mellor AL, Baban B, Chandler PR et al. Cutting edge: CpG oligonucleotides induce splenic CD19(+) dendritic cells to acquire potent indoleamine 2,3-dioxygenase-dependent T cell regulatory functions via IFN type 1 signaling [J]. Journal of Immunology 2005;175:5601-5605.
[114] Meneses KD, Mcnees MP. Upper extremity lymphedema after treatment for breast cancer: A review of the literature [J]. Ostomy Wound Management 2007;53:16-29.
[115] Meng YX, Carpentier AF, Chen L et al. Successful combination of local CpG-ODN and radiotherapy in malignant glioma [J]. International Journal of Cancer 2005;116:992-997.
[116] Milas L, Mason KA, Ariga H et al. CpG oligodeoxynucleotide enhances tumor response to radiation [J]. Cancer Research 2004;64:5074-5077.
[117] Munn DH, Mellor AL. The tumor-draining lymph node as an immune-privileged site [J]. Immunological review 2006; Oct;213:146-58
[118] McHutchison JG, Bacon BR, Gordon SC et al. Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis c virus [J]. Hepatology 2007;46:1341-1349.
[119] Molenkamp BG, van Leeuwen PAM, van den Eertwegh AJM et al. Immunomodulation of the melanoma sentinel lymph node: A novel adjuvant therapeutic option [J]. Immunobiology 2006;211:651-661.
[120] Molenkamp BG, van Leeuwen PAM, Meijer S et al. Intradermal CpG-B activatesboth plasmacytoid and myeloid dendritic cells in the sentinel lymph node of melanoma patients [J]. Clinical Cancer Research 2007;13:2961-2969.
[121] Mortarini R, Piris A, Maurichi A et al. Lack of terminally differentiated tumor-specific CD8(+) T cells at tumor site in spite of antitumor immunity to self-antigens in human metastatic melanoma [J]. Cancer Research 2003;63:2535-2545.
[122] Morganti AG, Cilla S, Valentini V et al. Phase I-II studies on accelerated IMRT in breast carcinoma: Technical comparison and acute toxicity in 332 patients [J]. Radiotherapy and Oncology 2009;90:86-92.
[123] National Cancer Institute, SEER Stat Fact Sheets: cancer of the Breast [R]. Bethesda: NCI, 2008
[124] Nathanson SD. Insights into the mechanisms of lymph node metastasis [J]. Cancer 2003;98:413-423.
[125] Nakano H, Yanagita M, Gunn MD. CD11c(+)B220(+)Gr-1(+) cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells [J]. Journal of Experimental Medicine 2001;194:1171-1178.
[126] Nagorsen D, Voigt S, Berg E et al. Tumor-infiltrating macrophages and dendritic cells in human colorectal cancer: relation to local regulatory T cells, systemic T-cell response against tumor-associated antigens and survival [J]. Journal of Translational Medicine 2007;5.
[127] Nichani AK, Kaushik RS, Mena A et al. CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep [J]. Cell Immunol. 2004; 227(1): 24-37
[128] Ning L, Fan XG, Chen ZH et al. Inhibition of the hepatitis B virus replication in vitro by an oligodeoxynucleotide containing cytidine-guano sine motifs [J]. Immunology Letters 2006;102:60-66.
[129] Nowroozalizadeh S, Jansson M, Adamsson J et al. Suppression of HIV replication in vitro by CpG and CpG conjugated to the non toxic B subunit of cholera toxin [J]. Current Hiv Research 2008;6:230-238.
[130] Olbrich ARM, Schimmer S, Heeg K et al. Effective postexposure treatment of retrovirus-induced disease with immunostimulatory DNA containing CpG motifs [J]. Journal of Virology 2002;76:11397-11404.
[131] Olbrich ARM, Schimmer S, Dittmer U. Preinfection treatment of resistant mice with CpG oligodeoxynucleotides renders them susceptible to friend retrovirus-induced leukemia [J]. Journal of Virology 2003;77:10658-10662.
[132] Opavsky A, Penninger J, Aitken K et al. Host susceptibility to CVB3 myocarditis in T cell knockout mice is associated with distinct myocardial cytokine expression repertoire [J]. Circulation 1999;100:429.
[133] Padalko E, Nuyens D, De Palma A et al. The interferon inducer ampligen [poly(I)-poly(C12U)] markedly protects mice against coxsackie 133 virus-induced myocarditis [J]. Antimicrobial Agents and Chemotherapy 2004;48:267-274.
[134] Pashenkov M, Goess G, Wagner C et al. Phase II trial of a Toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma [J]. Journal of Clinical Oncology 2006;24:5716-5724.
[135] Pasare C, Medzhitov R. Toll-like receptors: Linking innate and adaptive immunity. Mechanisms of Lymphocyte Activation and Immune Regulation X [J]: Innate Immunity 2005;560:11-18.
[136] Peng WM, Yu CF, Allam JP et al. Inhibitory oligodeoxynucleotides downregulate herpes simplex virus-induced plasmacytoid dendritic cell type I interferon production and modulate cell function [J]. Human Immunology 2007;68:879-887.
[137] Piccart M, Hamilton A. HER2/neu as a predictive factor in breast cancer [J]. European Journal of Cancer 1999;35:S84.
[138] Poindexter NJ, Sahin A, Hunt KK et al. Analysis of dendritic cells in tumor-free and tumor-containing sentinel lymph nodes from patients with breast cancer [J]. Breast Cancer Research 2004;6:R408-R415.
[139] Rayburn ER, Wang W, Zhang Z et al. Experimental therapy of prostate cancer with an immunomodulatory oligonucleotide: Effects on tumor growth, apoptosis, proliferation, and potentiation of chemotherapy [J]. Prostate 2006;66:1653-1663.
[140] Rayburn ER, Wang W, Zhang RW et al. Experimental therapy for colon cancer: Anti-cancer effects of TLR9 agonism, combination with other therapeutic modalities, and dependence upon p53 [J]. International Journal of Oncology 2007;30:1511-1519.
[141] Robinson NMK, Zhang HY, Bevan AL et al. Induction of myocardial nitric oxide synthase by Coxsackie B3 virus in mice [J]. European Journal of Clinical Investigation 1999;29:700-707.
[142] Rothenfusser S, Hornung V, Ayyoub M et al. CpG-A and CpG-Boligonucleotides differentially enhance human peptide-specific primary and memory CD8(+) T-cell responses in vitro [J]. Blood 2004;103:2162-2169.
[143] Rosset MB, Ballerini C, Gregoire S et al. Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice [J]. Journal of Immunology 2004;172:5168-5174.
[144] Sfondrini L, Besusso D, Rumio C et al. Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA [J]. Faseb Journal 2002;16:1749-1754.
[145] Shu S, Cochran AJ, Huang RR et al. Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy [J]. Cancer and Metastasis Reviews 2006;25:233-242.
[146] Speiser DE, Lienard D, Rufer N et al. Rapid and strong human CD8(+) T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909 [J]. Journal of Clinical Investigation 2005;115:739-746.
[147] Srinivasan S, Parsa V, Liu CY et al. Trastuzumab-induced hepatotoxicity [J]. Annals of Pharmacotherapy 2008;42:1497-1501.
[148] Sanders ME, Scroggins T, Ampil FL et al. Accelerated partial breast irradiation in early-stage breast cancer [J]. Journal of Clinical Oncology 2007;25:996-1002.
[149] Santin AD. Lymph node metastases - The importance of the microenvironment [J]. Cancer 2000;88:175-179.
[150] Saloustros E, Mavroudis D, Georgoulias V. Paclitaxel and docetaxel in the treatment of breast cancer [J]. Expert Opinion on Pharmacotherapy 2008;9:2603-2616.
[151] Sasse A, Wallich M, Ding Z et al. Coxsackie-and-adenovirus receptor mRNA expression in human heart failure [J]. Journal of Gene Medicine 2003;5:876-882.
[152] Sethi S, Lipford G, Wagner H et al. Postexposure prophylaxis against prion disease with a stimulator of innate immunity [J]. Lancet 2002;360:229-230.
[153] Sivori S, Carlomagno S, Moretta L et al. Comparison of different CpG oligodeoxynucleotide classes for their capability to stimulate human NK cells [J]. European Journal of Immunology 2006;36:961-967.
[154] Sun SQ, Zhang XH, Tough DF et al. Type I interferon-mediated stimulation of T cells by CgG DNA [J]. Journal of Experimental Medicine 1998;188:2335-2342.
[155] Schlaepfer E, Audige A, von Beust B et al. CpG oligodeoxynucleotides blockhuman immunodeficiency virus type 1 replication in human lymphoid tissue infected ex vivo [J]. Journal of Virology 2004;78:12344-12354.
[156] Scheller C, Ullrich A, McPherson K et al. CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells [J]. Journal of Biological Chemistry 2004;279:21897-21902.
[157] Schwaab T, Weiss JE, Schned AR et al. Dendritic cell infiltration in colon cancer [J]. Journal of Immunotherapy 2001;24:130-137.
[158] Scott LJ, Keam SJ. Letrozole: In postmenopausal hormone-responsive early-stage breast cancer [J]. Drugs 2006;66:353-362.
[159] Sohn WJ, Lee KW, Choi SY et al. CpG-oligodeoxynucleotide protects immune cells from gamma-irradiation-induced cell death [J]. Molecular Immunology 2006;43:1163-1171.
[160] Thompson JA, Kuzel T, Bukowski R et al. Phase lb trial of a targeted TLR9 CpG immunomodulator (CPG 7909) in advanced renal cell carcinoma (RCC) [J]. Journal of Clinical Oncology 2004;22:417S.
[161] Takahashi K, Nishikawa Y, Sato H et al. Dendritic cells interacting mainly with B cells in the lymphoepithelial symbiosis of the human palatine tonsil [J]. Virchows Archiv 2006;448:623-629.
[162] Takahashi K, Kenji A, Norihiro T et al. Morphological interactions of interdigitating dendritic cells with B and T cells in human mesenteric lymph nodes [J]. American Journal of Pathology 2001;159:131-138.
[163] Takeshita F, Gursel I, Ishii KJ et al. Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9 [J]. Seminars in Immunology 2004;16:17-22.
[164] Teleshova N, Kenney J, Jones J et al. CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting Simian immunodeficiency virus-specific T cells [J]. Journal of Immunology 2004;173:1647-1657.
[165] Teleshova N, Kenney J, Van Nest G et al. Local and systemic effects of intranodally injected CpG-C immunostimulatory-oligodeoxyribonucleotides in macaques [J]. Journal of Immunology 2006;177:8531-8541.
[166] Tsuge T, Yamakawa M, Tsukamoto M. Infiltrating dendritic/Langerhans cells inprimary breast cancer [J]. Breast Cancer Research and Treatment 2000;59:141-152.
[167] Ugolini C, Basolo F, Proietti A et al. Lymphocyte and immature dendritic cell infiltrates in differentiated, poorly differentiated, and undifferentiated thyroid carcinoma [J]. Thyroid 2007;17:389-393.
[168] Van der Stede Y, Verdonck F, Vancaeneghem S et al. CpG-oligodinucleotides as an effective adjuvant in pigs for intramuscular immunizations [J]. Veterinary Immunology and Immunopathology 2002;86:31-41.
[169] van der Most RG, Currie A, Robinson BWS et al. Cranking the immunologic engine with chemotherapy: Using context to drive tumor antigen cross-presentation towards useful antitumor immunity [J]. Cancer Research 2006;66:601-604.
[170] van Ojik HH, Bevaart L, Dahle CE et al. CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations [J]. Cancer Research 2003;63:5595-5600.
[171] Verthelyi D, Ishii KJ, Gursel M et al. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs [J]. Journal of Immunology 2001;166:2372-2377.
[172] Verthelyi D, Gursel M, Kenney RT et al. CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection [J]. Journal of Immunology 2003;170:4717-4723.
[173] Vincent IE, Lucifora J, Durantel D et al. Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro [J]. Antiviral Therapy 2009;14:131-135.
[174] Viale PH, Yamamoto DS. Cardiovascular toxicity associated with cancer treatment [J]. Clinical Journal of Oncology Nursing 2008;12:627-638.
[175] Vuylsteke RJCL, Molenkamp BG, Gietema HA et al. Local administration of granulocyte/macrophage colony-stimulating factor increases the number and activation state of dendritic cells in the sentinel lymph node of early-stage melanoma [J]. Cancer Research 2004;64:8456-8460.
[176] Vuylsteke RJCL, Molenkamp BG, van Leeuwen PAM et al. Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF-treated stage I melanoma patients is associated with high myeloid dendritic cell content [J].Clinical Cancer Research 2006;12:2826-2833.
[177] Vollmer J, Weeratna R, Payette P et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities [J]. European Journal of Immunology 2004;34:251-262.
[178] Wang H, Rayburn ER, Wang W et al. Immunomodulatory oligonucleotides as novel therapy for breast cancer: pharmacokinetics, in vitro and in vivo anticancer activity, and potentiation of antibody therapy [J]. Molecular Cancer Therapeutics 2006;5:2106-2114.
[179] Wang XJ, Bao MS, Wan M et al. A CpG oligodeoxynucleotide acts as a potent adjuvant for inactivated rabies virus vaccine [J]. Vaccine 2008;26:1893-1901.
[180] Wang XS, Sheng Z, Ruan YB,et al. CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma [J]. World J Gastroenterol. 2005, 11(8):1220-1224
[181] Wang YX, Da Cunha V, Vincelette J et al. Antiviral and myocyte protective effects of murine interferon-beta and -alpha 2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice [J]. American Journal of Physiology-Heart and Circulatory Physiology 2007;293:H69-H76
[182] Weeratna RD, Bourne LL, Sullivan SM et al. Combination of a new TLR9 agonist immunomodulator (CpG 7909) and paclitaxel for treatment of metastatic Lewis Lung Carcinoma (LLC) [J]. Journal of Clinical Oncology 2004;22:702S.
[183] Wernette CM, Smith BF, Barksdale ZL et al. CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation [J]. Vet Immunol Immunopathol. 2002; 84(3-4): 223-36
[184] Wingender G, Garbi N, Schumak B et al. Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO [J]. European Journal of Immunology 2006;36:12-20.
[185] Xiong ZM, Gharagozlou S, Vengco I et al. Effective CpG immunotherapy of breast carcinoma prevents but fails to eradicate established brain metastasis [J]. Clinical Cancer Research 2008;14:5484-5493.
[186] Yamamoto S, Yamamoto T, Kataoka T, et al. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity [J]. Journal ofImmunology 1992; 148(12):4072-6
[187] Yasuda K, Rutz M, Schlatter B et al. CpG motif-independent activation of TLR9 upon endosomal translocation of "natural" phosphodiester DNA [J]. European Journal of Immunology 2006;36:431-436.
[188] Yasuda K, Yu P, Kirschning CJ et al. Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways [J]. Journal of Immunology 2005;174:6129-6136.
[189] Yoshizawa H, Chang AE, Shu SY. Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells [J].Cancer Res. 1992 Mar 1;52(5):1129-36
[190] Yoshizawa H, Chang AE, Shu SY. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2 [J]. J Immunol. 1991 Jul 15;147(2):729-37
[191] Yu D, Putta MR, Bhagat L et al. Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction [J]. Antimicrobial Agents and Chemotherapy 2008;52:4320-4325.
[192] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction [J]. Nucleic Acids Research 2003;31:3406-3415.
[193]陈忠明,田少敏,阮康成。高压力制备柯萨奇病毒疫苗[J]。生物化学与生物物理学报,2001,33(1):128～130
[194]李锋,张立红,田淑芝。干扰素治疗小儿病毒性心肌炎的临床和免疫学观察[J]。医学综述,2008,14(12):1915-1917。
[195]刘文春,黄星原。黄芪注射液对病毒性心肌炎小鼠生存率及预后的影响[J],湖北中医杂志,2004,26(7):10-11
[196]王雪峰,郭津津,魏克伦等。小柴胡汤分解剂抗柯萨奇B3病毒感染及其对心肌炎防治作用的研究[J],中国医科大学学报,2000,29(2):115-117
[197]徐海燕,马沛然。西洋参对小鼠病毒性心肌炎的疗效及机制[J],山东中医药大学学报,2002,26(6):458-461