多发性骨髓瘤的EBV、B7—1及主动免疫治疗研究

目的：探讨EB病毒在多发性骨髓瘤的病因作用及新型疫苗治疗多发性骨髓瘤的作用。方法：用多聚酶链反应（PCR）的方法检测21例多发性性骨髓瘤（MM）患者瘤细胞的EB病毒DNA,应用间接免疫荧光法检测瘤细胞上MHCⅠ、Ⅱ类分子及共刺激分子B7－1的表达,应用细胞因子与瘤细胞制成的疫苗主动免疫治疗4例MM患者,结果：发现66．7％的MM患者瘤细胞内有EB病毒DNA。瘤细胞膜多表达MHCⅠ、Ⅱ分子及B7－1。4例MM患者用疫苗治疗后3例取得明显疗效。结论EB很病毒很可能是MM的病因,与B7－1阳性的细胞及细胞因子联合制成的疫苗主动免疫治疗MMV具有较明显的疗效。     

独特型蛋白致敏的树突细胞瘤苗治疗多发性骨髓瘤

多发性骨髓瘤(MM)有独特型蛋白,为肿瘤特异性抗原.大量研究表明,使用负载独特型蛋白的树突细胞(DC)瘤苗进行主动免疫治疗,有助于消除(MM)微小残留病变,延长患者的无病生存期.近年来在抗肿瘤免疫治疗中的应用已成为肿瘤主动特异性免疫治疗的研究热点.     

多发性骨髓瘤的免疫治疗研究进展

免疫治疗是最有希望控制和清除微小残留病(MRD)从而治愈多发性骨髓瘤(MM)的治疗方法。动物模型和临床实验研究显示MM的免疫治疗尤其是以免疫接种为基础的主动免疫具有巨大的应用潜力。对3种主要的免疫接种策略：肿瘤特异性抗原疫苗、DC树突细胞介导的免疫治疗和瘤苗在MM治疗中的研究进展加以综述。     

急性白血病的新型主动免疫治疗研究

目的：应用化疗不能治愈大多数急性白血病，近年人们对基因工程疫苗寄以厚望，动物实验已展现其具有明显疗效，但迄今尚未见单独应用白血病疫苗主动免疫及治疗急性白血病患取得明显疗效的报道。本应用基因工程生产的细胞因子及白血病细胞制备的疫苗，主要免疫治疗21例急性白血病，以探讨提高主动免疫治疗急必白血病疗效的方法。方法：1．新型白血病疫苗制备；取化疗前或化疗末缓解的白血患自身骨髓15ml肝素抗凝，应用淋巴细胞分离液分离出富含白血病细胞的骨髓单个核细胞，经丝裂霉素处理后，加入含基因工程生产的粒单细胞集落刺激因子，白细胞介素2及白细胞介素6等的福氏佐剂中。2．主动免疫治疗程序：对所有接受免疫治疗患在停用化疗两周后进行骨髓检查并开始接种疫苗，每次用含10^8个白血病细胞的疫苗1．6ml，分两点前臂皮下注射，每隔7天重复一次，共4次，然后长期随访。如无效时再改用其它治疗；如有效，每隔6个月加强注射一次。结果：21例急性白血病患均因化疗无效或不能耐受耐改用疫苗治疗。免疫治疗达到完全缓解（CR）5例，为24％，部分缓解（PR）4例，为19％，微效（MR）3，为14％，总有效率达到575。有效多为女性，低增生性急性单核细胞白血病（M5）。疗效平均持续时间约7个月后易复发；而例有效率患在每6个月时应用此疫苗加强免疫治疗，已分别存活18个月及24个月。用此疫苗治疗时未见明显毒副作用，对5例疫苗治疗有效及3例无效患应用免疫组化方法检测其白血病细胞膜上MHC1．2类分子及共刺激分子（B7－1），发现5例有效病例的MHC1．2类分子及B7－1均为阳性，而3例无效病例虽MMHC1．2类分子阳性，但B7分子均为阴性。结论：结果表明应用此新型白血病疫苗主动免疫治疗化疗无效及不能耐受化疗的某些急性白血病，具有肯定疗效，且经济、简便、无明显毒副作用，值得进一步研究，特别适用于单核细胞性的、B7阳性的、低增生性的白血病患。     

多发性骨髓瘤抗独特型决定簇免疫治疗研究进展

传统疗法通常无法治愈多发性骨髓瘤(MM),近年来提出了用于提高长期生存率的免疫治疗方法.其中,对于已经接受大剂量化疗以及自体或异体干细胞移植的MM患者,通过使用树突细胞(DC)介导的抗独特型决定簇瘤苗进行免疫治疗的体内研究结果证实,独特性抗肿瘤效应的产生能够有效根除微小残留病变,明显延长患者的无病生存期.现综述MM抗独特型决定簇(Id)治疗性瘤苗、细胞瘤苗以及基因疫苗的相关临床和临床前研究进展.     

多发性骨髓瘤抗独特型决定簇免疫治疗研究进展

传统疗法通常无法治愈多发性骨髓瘤(MM)，近年来提出了用于提高长期生存率的免疫治疗方法。其中，对于已经接受大剂量化疗以及自体或异体干细胞移植的MM患者，通过使用树突细胞(DC)介导的抗独特型决定簇瘤苗进行免疫治疗的体内研究结果证实，独特性抗肿瘤效应的产生能够有效根除微小残留病变，明显延长患者的无病生存期。现综述MM抗独特型决定簇(Id)治疗性瘤苗、细胞瘤苗以及基因疫苗的相关临床和临床前研究进展。     

树突状细胞融合瘤苗在肿瘤免疫中的研究进展

树突状细胞(DC)是诱导初始免疫应答反应的专职抗原提呈细胞,它能捕获抗原并加工处理成小分子多肽,通过MHC Ⅰ类和Ⅱ类分子提呈给T细胞和B细胞.Dc与肿瘤细胞融合所获得的融合瘤细胞,在共刺激信号存在的条件下,能够加工处理细胞内许多已知和未知的肿瘤相关抗原(TAA),并通过MHC Ⅰ类和Ⅱ类分子提呈.目前,DC融合瘤苗在动物和临床研究中取得了许多进展,已成为肿瘤免疫治疗的热点之一.     

骨髓微环境与多发性骨髓瘤

 骨髓微环境不仅支持正常造血细胞的生长和分化,而且有助于肿瘤细胞的生长,它在多发性骨髓瘤细胞的增生、分化、凋亡中起重要作用。多发性骨髓瘤（MM）的瘤细胞定居在骨髓很少浸润骨髓以外的器官,与肿瘤细胞所处的骨髓造血微环境密切相关。骨髓瘤细胞与骨髓基质细胞及细胞外基质相互黏附,并产生细胞因子,进而影响肿瘤细胞的存活,而且还影响瘤细胞对治疗的反应。MM是一个进展性、致死性疾病,传统的化疗很易发生耐药。目前随着免疫学、分子生物学的发展及对骨髓微环境的研究,一些新型的以生物学为基础的药物不仅针对瘤细胞本身,而且以骨髓微环境为靶子,通过改变骨髓微环境而达到克服耐药的目的,从而延长骨髓瘤患者的无病生存期。     

疫苗治疗多发性骨髓瘤的研究进展

多发性骨髓瘤(multiple myeloma,MM)是一种浆细胞恶性肿瘤,约占血液系统恶性肿瘤的10％.传统大剂量化疗及自体干细胞移植提高了患者的生存率,近十年来新型药物的出现也极大地延长了患者的OS;然而,化疗毒性作用及移植相关死亡率也不容忽视.在现有的治疗手段下,大部分MM患者最终会复发耐药而死亡,因此亟需寻找新的治疗方法.肿瘤免疫治疗在控制骨髓瘤方面有极好的前景,可能为MM患者提供除传统化疗之外的新的治疗选择.治疗性疫苗可通过诱导免疫应答以清除骨髓瘤细胞,大量研究着眼于研发可产生骨髓瘤特异性免疫的肿瘤疫苗,目前许多疫苗正处于临床试验阶段,并已经开始呈现出令人欣喜的结果.本文就MM疫苗的最新主要研究进展进行综述.     

MUC1黏蛋白在多发性骨髓瘤中的表达及其意义

目的探讨MUC1黏蛋白分子在多发性骨髓瘤患者中的表达、意义及在肿瘤生物治疗中的应用前景。方法应用免疫细胞化学S蛳P法检测了9例多发性骨髓瘤患者的骨髓中MUC1黏蛋白基因的表达情况。结果多发性骨髓瘤(MM)患者MUC1阳性表达明显高于对照组,两组差异有显著性(P<0.001)。结论MUC1黏蛋白在多发性骨髓瘤中以非糖基化的形式表达异常增高,其表达与MM的病情进展及预后可能相关。MUC1免疫原可作为疫苗应用于多发性骨髓瘤生物治疗的研究。     

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.     

Limiting dilution cloning of B cells from patients with multiple myeloma: emergence of non-malignant B-cell lines

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of slowly proliferating malignant plasma cells in the bone marrow (BM). Several reports have shown the existence of an abnormal B-cell compartment including proliferative idiotypic B cells (i.e., B cells bearing the same idiotypic determinants as the myeloma protein) in the BM and peripheral blood (PB) of patients with MM. In order to study whether this abnormal compartment can be grown in vitro, we cultured the PB and BM of 23 patients with MM using limiting dilution methods. Our purpose was to restrict the effect of suppressor cells and the possible overgrowth of the cultures by the more rapidly growing B cells, which occurs in bulk cultures. Spontaneously growing cells were obtained only from patients seropositive for the Epstein-Barr virus (EBV) and all the cultures were composed of B cells carrying the EBV genome. Thus, positive cultures were generated only in the presence of B cells latently infected with EBV in vivo. The mean frequency of these B cells (1 in 25,000 B cells) was as low in MM patients as in healthy donors. This low frequency indicated that malignant cells do not bear the EBV genome in vivo and that the in vivo regulation of the EBV infection is unaffected in patients with MM. No Ig-gene rearrangements, specific of the autologous myeloma cells, were found in the cell lines obtained from BM or PB. Thus, the putative malignant B cells or myeloma cells were not able to generate cell lines in vitro, either spontaneously or after endogenous infection with EBV.     

Elevated Th22 as well as Th17 cells associated with therapeutic outcome and clinical stage are potential targets in patients with multiple myeloma

T helper (Th) cell imbalance plays important roles in tumor development and their effects in Multiple myeloma (MM) remain unclear. In the present study, we investigated the levels and clinical significance of Th22, Th17 and Th1 cells in patients with MM. Th subsets were examined by flow cytometry. Plasma IL-22, IL-17A and IFN-γ concentrations were measured by ELISA. AHR and RORC mRNA expression was examined by RT-PCR. Here, we found that the frequency of Th22 cells was significantly elevated in peripheral blood (PB) and bone marrow (BM) of newly-diagnosed MM patients, and recovered in complete remission patients after chemotherapy. The circulating Th17 cells accompanied by IL-17A levels were also up-regulated in MM patients and decreased after remission. We also found that there was a significantly positive correlation between Th22 and Th17 cells in MM patients. Moreover, the frequencies of Th22 and Th17 cells were higher in stage III than in stage I+II of MM. Our data demonstrated that Th22 and Th17 cells might be important therapeutic targets in multiple myeloma and could facilitate the effect of antitumor immunotherapy.     

Humanized anti-interleukin-6 receptor monoclonal antibody induced apoptosis of fresh and cloned human myeloma cells in vitro

We investigated the effect of anti-IL-6 receptor monoclonal antibody (hPM1) on the in vitro proliferation of cloned and freshly isolated myeloma cells from 20 patients with advanced stage multiple myeloma (MM). Humanized PM1 significantly inhibited the growth of a myeloma cell line in a dose-dependent manner and inhibited more than 30% of the proliferation of fresh myeloma cells in 10 of the 19 cases. Flow cytometric analysis using annexin V and 7AAD showed that hPM1 induced apoptosis of myeloma cells. These observations suggest the possibility of using hPM1 for treating some patients with MM whose growth depends on IL-6.     

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.     

Dendritic cell-based immunotherapy in multiple myeloma

Most patients with multiple myeloma (MM) cannot be cured with currently available therapies. Although complete remission could be achieved in about 50% of newly diagnosed patients with high-dose chemotherapy and tandem transplantation, relapses of the underlying disease occur frequently. To realize long-term disease-free survival, it will be necessary to develop complementary therapies that are non-cross-resistant with chemotherapy. To this end, immunotherapy aimed at inducing or enhancing tumor-specific immunity that may control or eradicate remaining tumor cells may be an appealing method. Dendritic cells (DCs) are professional antigen-presenting cells and considered the best natural adjuvants for immunotherapy in malignancies. Vaccination with tumor antigen-pulsed DCs has been shown to be protective and therapeutic in animal tumor models, and induced a strong tumor-specific immunity and durable tumor regression in human solid tumors and B-cell lymphoma. As a result, clinical trials in various human malignancies have been initiated. This review will focus on DC-based immunotherapy in MM. I will discuss myeloma antigens and antigen-specific immune responses, the capacity of DCs to present myeloma antigens and induce cytotoxic T-cell responses, and clinical experience of DC vaccination in myeloma patients.     

The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis.