Transfer factor (TF) treatment of patients with HBs-Ag-positive chronic active hepatitis

Summary It is a clinically and experimentally well supported working hypothesis that infection with hepatitis B virus may result in chronic active hepatitis in patients with suspected immune deficiencies. On this basis, a pilot study was performed in order to evaluate the effect of specific transfer factor (TF) in the treatment of HB_s-Ag-positive chronic active hepatitis. From the leukocytes of 500 ml venous blood each of 40 volunteers that had completely recovered from acute virus hepatitis B within the last 6 months, a unique TF pool (40 units of TF) was prepared according to the method of Lawrence. Preexaminations indicated that this preparation was able to enhance cellular immune reactions in vitro. Thirteen patients with HB_s-antigenemia and chronic active hepatitis (i.e., two liver biopsies within the last 6 or more months with the histological criteria of chronic aggressive hepatitis according to de Groote, elevated serum levels of bilirubin, alkaline phosphatase, transaminase activities, and/or -globulines) were randomized: Seven received s.c. injections of two units of TF each on days 1 and 15, the other six saline. Conversion of skin reactions to some ubiquitous antigens occurred in the TF group, but no significant and constant drop of HB_s-Ag serum titers was observed. Although some of the biochemical parameters seemed to ameliorate in the TF group, the differences versus the control group did not prove to be significant within the limited number of patients under observation. The in vitro reactivity of patients' lymphocytes to HB_s-Ag, tested by means of the³H-thymidine uptake, was never found enhanced after TF application. In the used doses, specific TF was not effective in the treatment of HB_s-Ag-positive chronic active hepatitis; unfavorable side-effects were not observed.     

转染肿瘤总RNA的DC疫苗诱导抗肝癌特异性免疫

目的：探讨转染人肝癌总RNA的树突状细胞(DC) 疫苗体外诱导特异性细胞毒性T淋巴细胞(CTL)的作用。 方法： 采用原发性肝癌（HCC）病人外周血单核细胞（PBMC），在粒/巨细胞集落刺激因子(GM-CSF)和白细胞介素-4(IL-4) 刺激下增殖分化为DC细胞；从人肝癌细胞中体外扩增肝癌RNA。以HCCRNA转染DC细胞，并与PBMC混合培养诱导扩增CTL。MTT法测定CTL的杀瘤活性。 结果： 转染HCCRNA 48 h后， DC表面分子CD83、CD86和HLA-DR表达明显增高。转染HepG-2细胞HCCRNA的DC和病人HCCRNA诱导的CTL对HepG-2细胞和病人HCC细胞的杀瘤活性均明显高于正常肝细胞RNA+DC、脂质体+DC、Opti-MEM+DC以及空白对照组；而对胃癌SGC-7901细胞无杀伤活性。 结论： 以肝癌RNA为肿瘤抗原，DC作为疫苗的抗原提呈细胞，体外冲击致敏DCs，能诱导肝癌特异性CTL。本研究为HCC术后复发和转移的防治提供一种可能有效的疫苗治疗方法。     

IL-18基因修饰增强肿瘤抗原多肽致敏的树突状细胞体内诱导的抗肿瘤免疫反应

目的 :研究肿瘤抗原多肽致敏的白细胞介素 18(IL 18)基因修饰的树突状细胞体内诱导的抗肿瘤免疫反应。方法 :①以Lewis 3LL肺癌细胞特异性抗原肽mut1冲击致敏IL 18基因修饰的骨髓来源的树突状细胞 (DC IL 18 mut1) ,每次用其 1× 10 5 只皮下免疫小鼠 2次 ,然后测定脾细胞的NK活性及CTL杀伤活性 ;②以DC IL 18 mut1每次 2× 10 5 只皮下免疫 1次 ,然后再以 5× 10 53LL细胞攻击 ,在诱导及效应阶段分别以单抗阻断不同免疫成份 ,观察肿瘤的生长。结果 :以DC IL 18 mut1皮下免疫后可诱导出比DC mut1等免疫组更高水平的 3LL肺癌细胞特异性CTL ,并使NK活性明显增加 ;单抗体内阻断实验提示在DC IL 18 mut1免疫诱导阶段 ,CD4 + T细胞和抗原共刺激分子、IFN γ均起到重要作用 ,而效应阶段CD8+ T、IFN γ、NK起作用 ,而CD4 + T则是非必需的。结论 :DC IL 18 mut1皮下免疫后可诱导高水平的抗肿瘤免疫活性 ,其机理与抗原有效提呈、特异性CTL诱导、NK活性增加以及CD4 + 、CD8+ T、NK细胞、IFN γ参与密切相关。     

CD20scFv-IgGFc T 淋巴细胞的建立及其对B细胞淋巴瘤靶向杀伤作用的比较

目的：研究CD20scFv嵌合T淋巴细胞靶向杀伤Daudi细胞时杀伤的效果和T细胞活化情况。方法：将两种质粒转染至PA317细胞中，用转染成功的PA317上清液感染外周血T淋巴细胞后经800 mg/L的G418筛选1周后去杀伤Daudi、K562细胞，分别在不同时点用流式细胞仪检测Daudi细胞AnnexinⅤ的阳性率 ，用ELISA检测细胞因子IL-2、IFNγ。结果：Daudi细胞AnnexinⅤ的阳性率在24 h内两实验组与K562组相比明显增高，而实验组之间没有显著差异。72 h时CD20scFv-IgGFc-CD28-ζ组比CD20scFv-IgGFc组IL-2（1 509.00 ng/L比220.54 ng/L）和IFNγ（912.16 ng/L比251.42 ng/L）的分泌量有显著增高。结论：①两种CD20scFv特异的T细胞在引起Daudi细胞早期凋亡无显著差异，说明在CD20scFv的靶向杀伤过程中CD28-ζ基因可能不主导Daudi细胞的早期凋亡。②CD20scFv-IgGFc-CD28-ζ组IL-2、IFNγ增幅更加明显，说明CD3ζ和CD28嵌合的T细胞可以自身活化而不受MHC的限制，从而增强了T细胞的活化和杀伤等功能。     

Protection from experimental autoimmune diabetes in the non-obese diabetic mouse with soluble interleukin-1 receptor

We have evaluated the effects of a treatment with soluble interleukin-1 receptor (sIL-1R) in the accelerated model of autoimmune diabetes induced by cyclophosphamide (CY) in the non-obese diabetic (NOD) mouse. Prior to the CY challenge (350 mg/kg body weight), female euglycemic NOD mice were randomly divided into three groups (A–C). Groups B and C were treated daily from 1 day before to 13 days after the CY challenge with sIL-1R at doses of 0.2 and 2 mg/kg body weight. Group A was treated with PBS. By 2 weeks after CY administration, an acute form of autoimmune diabetes with glycosuria, hyperglycemia and severe insulitis occurred in the majority (13/20, 65%) of the control mice (group A). In contrast, repeated injections with sIL-1R protected NOD mice from insulin-dependent diabetes mellitus (IDDM) development in a dose-dependent fashion; the incidence of IDDM was 53.3% (8/15) in the mice treated with 0.2 mg/kg and only 6.7% (1/15) in those treated with 2 mg/kg. However, none of the doses of the sIL-1R reduced the extent of insulitis in NOD mice. Importantly, the anti-diabetogenic property of sIL-1R may not involve major T cell function impairment; accordingly, in parallel experiments, splenic lymphoid cells from NOD mice not challenged with CY, but treated with 2 mg/kg sIL-1R for 5 consecutive days showed a normal distribution of mononuclear cell subsets and maintained their capacity to secrete interferon-γ and IL-2 and to proliferate in response to polyclonal mitogenic stimulation with concanavalin A.     

Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor-immunoglobulin fusion proteins

Tumor necrosis factor (TNF) activity was inhibited during the development of actively-induced, chronic relapsing experimental allergic encephalomyelitis (CREAE) in Biozzi AB/H mice, using a mouse TNF-specific (TN3.19.12) antibody and bivalent human p55 and p75 TNF receptor-immunoglobulin (TNFR-Ig) fusion proteins. The development of disease could be inhibited when repeated doses of antibody were administered prior to the anticipated onset. It has now also been shown that a therapeutic effect is evident even when antibody is administered after the onset of clinical signs, further indicating an important role for TNF in pathogenic effector mechanisms in CREAE. Although biologically-active TNF was not detected in the circulation, TNF-α was detected in lesions within the central nervous system (CNS). This suggested that the CNS may be the main site for TNF-specific immunomodulation and was supported by the observation that intracranial injection was significantly more potent than that administered systemically, for both antibody and TNFR-Ig fusion proteins. The fusion proteins were as effective as antibody at doses 10—100-fold lower than that used for antibody, reflecting their higher neutralizing capacity in vitro. Although treatment was not curative and relapse inevitably occurred in this model if treatment was not sustained, the data indicate that anti-TNF immunotherapy, especially within the CNS, can inhibit CREAE and may, therefore, be useful in the control of human neuroimmunological diseases.     

Targeted cytokines for cancer immunotherapy

Targeting of cytokines into the tumor microenvironment using antibody-cytokine fusion proteins, called immunocytokines, represents a novel approach in cancer immunotherapy. This article summarizes therapeutic efficacy and immune mechanisms involved in targeting interleukin-2 (IL-2) to neuroectodermal, tumors using ganglioside GD2-specific antibody-IL-2 fusion protein (ch14.18-IL-2). Treatment of established melanoma metastases with ch14.18-IL-2 resulted in eradication of disease followed by a vaccination effect protecting mice from lethal challenges, with wild-type tumor calls. In a syngeneic neuroblastoma model, targeted IL-2 was effective in the amplification of a weak memory immune response previously induced by IL-12 gene therapy using an engineered linear version of this heterodimeric cytokine. These findings show that targeted IL-2 may provide an effective tool in cancer immunotherapy and establish the missing link between T cell-mediated, vaccination and objective clinical responses.     

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210^bcr-abl fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210^bcr-abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210^bcr-abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.     

用糖基化磷脂酰肌醇B7-1锚定肿瘤细胞膜进行免疫治疗

目的 研制抗肿瘤免疫治疗的新疫苗.方法 用蛋白转化法将B7-1锚定在肿瘤细胞膜上,免疫C57BL-6小鼠后,一组小鼠取脾细胞进行T细胞扩增和细胞毒T淋巴细胞(CTL)功能检测,另一组小鼠进行肿瘤细胞接种试验.结果 用GPI-B7-1修饰的肿瘤细胞膜免疫小鼠后诱发了肿瘤特异性T细胞扩增和CTL.且该疫苗有一定的保护小鼠免受肿瘤细胞侵袭的作用.结论 GPI蛋白转化法为人类抗肿瘤免疫治疗提供了新的、有效的修饰肿瘤细胞膜的方法.     

Study of the protective effects of hyperimmune immunoglobulins G and M against endotoxin in mice and rats

We prepared solutions of human IgM and IgG to various lipopolysaccharide (LPS) species. These were then tested, along with solutions of non-LPS specific human IgG or IgM, for their ability to confer passive immunity against experimental endotoxemia in two animal models. The immunoglobulins were first tested for an effect on the lethality induced by seven different LPSs in actinomycin-D sensitized mice, or by three different bacteria in normal mice. When the immunoglobulins were administered 1 h before challenge, a small protective effect was observed. This protection was dependent upon both the anti-LPS agent, the chemical composition of the LPS, or the strain of Gram-negative bacteria used for injection. The anti-LPS IgM and IgG preparations reduced the mortality induced by Escherichia coli but not by Serratia marcescens or Klebsiella pneumoniae, indicating protection by strain-specific antibodies. When the antibodies were preincubated with LPS or bacteria for 30 min before administration, almost complete protection was seen. The influence of these immunoglobulin preparations or of human albumin (as a control) on the hypotensive and vascular-permeabilizing effects of LPS in rats was then studied. A dose-dependent inhibitory effect was observed with IgG preparations and albumin. At 200 mg/kg, anti-LPS IgG reduced the effects of LPS, while at 400 mg/kg, both anti-LPS and normal IgG preparations showed protection, as did human albumin used at the same dose. The IgM-enriched preparation worsened the initial hypotensive phase after LPS, whereas the anti-LPS IgM significantly reduced the second phase of the hypotension, but only at the largest dose of 400 mg/kg. In this second model using the rat, a clear difference between the activity of IgG and IgM was thus observed. We conclude that pretreatment with human immunoglobulins from large plasma pools modestly, but significantly, attenuated the effects of murine and rat Gram-negative sepsis, but that protection was incomplete. Our results suggest that single regimen intervention strategies may not be sufficient to influence the course of the disease. Received: 12 December 1998