Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients

The generation of T lymphocytes with specific reactivity against tumor antigens is a prerequisite for effective adoptive transfer therapies. Melanoma-specific lymphocyte cultures can be established from tumor infiltrating lymphocytes (TILs) by in vitro culture in high levels of IL-2. We have optimized methods for generating melanoma-reactive TIL cultures from small resected tumor specimens. We report a retrospective analysis of 860 attempted TIL cultures from 90 sequential melanoma biopsy specimens from 62 HLA-A2+ patients. Multiple independent TIL derived from a single tumor often exhibited substantial functional and phenotypic variation. Tumor specific activity was detected in TIL from 29 (81%) of 36 patients screened. TIL cultures selected for high activity were generally capable of large numerical expansion using a single round of a rapid expansion protocol. Limited clonal T-cell populations in an oligoclonal TIL culture could confer specific tumor recognition in these highly selected, highly expanded TIL cultures. These methods were efficient at generating TILs suitable for adoptive transfer therapy.     

利妥昔单抗联合二线方案治疗七例复发难治性霍奇金淋巴瘤患者疗效观察

目的 探讨利妥昔单抗联合二线方案治疗复发难治性霍奇金淋巴瘤(HL)患者的疗效及安全性.方法 7例复发难治性HL患者中2例接受R-GDP(E)[利妥昔单抗、吉西他滨、顺铂、地塞米松、(依托泊苷)]方案治疗,2例接受R-IGVP(利妥昔单抗、异环磷酰胺、吉西他滨、长春瑞滨、泼尼松)方案治疗,3例接受R-BEACOPP(利妥昔单抗、博来霉素、依托泊苷、多柔比星、环磷酰胺、长春新碱、甲基苄肼、泼尼松)方案治疗.观察患者治疗过程中及其后的不良反应和疗效.结果 7例患者中男3例,女4例,发病时中位年龄21(12～36)岁;结节性淋巴细胞为主型HL(NLPHL)1例,经典型HL 6例(包括4例结节硬化型,1例淋巴细胞为主型和1例混合细胞型).中位疗程数为4(1～4)个,中位随访时间为29(24～58)个月.7例患者中4例达完全缓解,2例达疾病稳定,1例死亡.2年总生存率为85.7％.不良反应均可耐受,以骨髓抑制为主.5例患者化疗后行外周血自体造血干细胞移植治疗.结论 利妥昔单抗联合二线方案治疗复发难治性HL患者安全、有效.     

Requirements for the induction and adoptive transfer of cyclosporine- induced syngeneic graft-versus-host disease

These studies further delineate the requirements for the establishment and transfer of SGVHD. We show that (a) two mechanisms distinguishable by radiation and drug sensitivities exist, (b) lethal irradiation correlates with a 100% incidence in the induction of SGVHD, whereas (c) both sublethal or lethal irradiation and cytoxan therapy are effective in ablating the host autoregulatory system in order to transfer autoreactivity, (d) unfractionated as well as nylon wool-nonadherent splenocytes effectively inhibit the transfer of autoimmunity, and (e) OX19 depletion of that population, however, destroys the autoregulatory effect present in normal splenocytes. To demonstrate complete inhibition of immune reactivity, twice the number of unfractionated splenocytes from normal animals was required for every splenocyte from autoimmune donors. Last, the infusion of effector splenocytes on 4, 7, and 14 d after transplantation correlates to a decrease from 100%, 70 to 0% incidence of SGVHD, thus emulating the incidence obtained in a pretransplant rat within 2 wk. These findings further clarify the immunobiological complexity of SGVHD and suggest that since autoregulatory cells already exist in normal animals that CsA-induced autoimmunity is a reflection of not an induced reactivity specific to one therapeutic reagent but the uncoupling of normal immunologic mechanisms essential in controlling autoimmunity.     

Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation

Adenovirus infection after allogeneic hematopoietic stem cell transplantation is still causing significant morbidity and mortality, especially in children. It has been demonstrated that a sufficient host T-cell response is essential to clear the virus. Adoptive transfer of specific T-cell immunity from the donor to the recipient has become a new treatment option for patients with systemic adenoviral infection who lack specific T-cell responses. The adenoviral hexon protein was shown to be an immunodominant T-cell target. We describe here a Good Manufacturing Practice-compatible generation of hexon-specific T cells developed by isolating interferon-gamma-secreting T cells after stimulation of mononuclear cells ex vivo with hexon protein. Phenotypical and functional characterization of the generated, specific T-cell product resulted in a mixed population of CD4 and CD8-positive T cells with an intermediate effector memory phenotype. Isolated hexon-specific T cells showed high expansion potential in vitro and specific cytotoxicity. T-cell lines, directed against type 5 hexon protein showed good crossreactivity against viral strains from other adenovirus species. The availability for isolation of hexon-specific T cells among 76 hematopoietic stem cell transplantation donors showed in > 72% a sufficient T-cell response (0.05% of T cells). In conclusion, Good Manufacturing Practice-grade selection of adenovirus-specific T cells for adoptive immunotherapy by hexon-induced secretion of interferon-gamma has been established. Adoptive T-cell transfer could potentially restore T-cell immunity against adenovirus after allogeneic stem cell transplantation.     

Brentuximab vedotin联合氮芥治疗六例复发难治性霍奇金淋巴瘤疗效分析

目的 观察Brentuximab vedotin (BV)联合盐酸氮芥治疗复发难治性经典型霍奇金淋巴瘤(cHL)患者的疗效及不良反应.方法 对6例BV单药治疗失败后cHL患者给予BV联合盐酸氮芥治疗,具体用药:BV 1.2～1.8 mg/kg第1天静脉滴注,盐酸氮芥6 mg/m2第1天静脉滴注,每21d为1个疗程,中位疗程数为4(3～8)个.治疗后每2个疗程行CT检查,每4个疗程行PET-CT检查,每个疗程前行血常规和生化检查,评价临床疗效与不良反应.结果 6例患者中男4例,女2例,中位年龄20.5(17～26)岁;2例为BV治疗达完全缓解后复发,4例为BV治疗后病情稳定.中位观察时间为15(11～25)周.6例患者接受BV联合盐酸氮芥治疗后2例获得完全缓解,4例获得部分缓解,总有效率为100％.4例患者出现骨髓抑制(Ⅰ度2例,Ⅳ度2例);2例出现Ⅱ度胃肠道反应;1例出现Ⅰ度肝损伤,心肌酶增高;1例出现Ⅰ度口腔溃疡.不良反应可耐受或对症处理后症状缓解,未影响患者治疗.结论 BV联合盐酸氮芥治疗BV单药治疗失败后复发难治性cHL,疗效显著,不良反应可耐受.     

The use of osteopathic manipulative treatment as adjuvant therapy in patients with peripheral arterial disease

Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis associated with impaired endothelial function and intermittent claudication is the hallmark symptom. Hypothesizing that osteopathic manipulative treatment (OMT) may represent a non-pharmacological therapeutic option in PAD, we examined endothelial function and lifestyle modifications in 15 intermittent claudication patients receiving osteopathic treatment (OMT group) and 15 intermittent claudication patients matched for age, sex and medical treatment (control group). Compared to the control group, the OMT group had a significant increase in brachial flow-mediated vasodilation, ankle/brachial pressure index, treadmill testing and physical health component of life quality (all p<0.05) from the beginning to the end of the study. At univariate analysis in the OMT group there was a negative correlation between changes in brachial flow-mediated vasodilation and IL-6 levels (r=-0.30; p=0.04) and a positive one between claudication pain time and physical function score (r=0.50; p=0.05). In conclusion, despite the relatively few patients in our study, these results suggest that OMT significantly improves endothelial function and functional performance in intermittent claudication patients along with benefits in quality of life. This novel treatment combined with drug and lifestyle modification might be an effective alternative to traditional training based on exercise.     

Donor-derived b2a2-specific T cells for immunotherapy of patients with chronic myeloid leukemia

The BCR-ABL fusion proteins, b2a2 and b3a2, are potential targets for a beneficial graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation for chronic myeloid leukemia (CML). This study demonstrates that CD4+ T cells specific to the b2a2 peptide can be generated from a normal allogeneic stem cell transplant donor after stimulation with monocyte-derived dendritic cells (Mo-DC) using culture conditions applicable to clinical use. Stimulation of donor T-cell enriched mononuclear cells (MNC) with b2a2-pulsed Mo-DC produced approximately 3 x 10(9) b2a2-specific CD4+ T cells. The CD4+ T cells were HLA-DR7 restricted. These results confirm that the generation of donor derived b2a2-specific T cells for clinical use is feasible and warrants clinical testing after stem cell transplantation.     

Demonstration and characterization of immunosuppressive factors in sera from patients with Crohn's disease

The effect of sera from 17 patients with Crohn's disease, 8 with ulcerative colitis or 5 with intestinal tuberculosis on the proliferative response of mouse spleen cells induced by phytohemagglutinin (PHA) was studied. Sera from patients with Crohn's disease markedly suppressed the blastogenesis of mouse spleen cells (S.I. = 6.8 +/- 2.0, % suppression = 83%), as compared with normal sera (S.I. = 41.0 +/- 5.2, p less than 0.001, % suppression = 0). Conversely, ulcerative colitis sera did not suppress the blastogenesis of mouse spleen cells (S.I. = 43.5 +/- 8.7, % suppression = -6%), nor the sera of intestinal tuberculosis (S.I. = 38.9 +/- 4.0, % suppression = 6%). Thus, we confirmed the possible existence of immunosuppressive factors in Crohn's disease. Moreover, immunosuppressive factors in Crohn's disease were characterized for biochemical properties. The approximate molecular weight is 45,000 estimated by diafiltration and gel filtration on a Sephadex G-75 column. Analytical isoelectric focusing showed an increased amount of acidic protein in fractionated sera (m.w. ranging 30,000-50,000) from patients with Crohn's disease and ulcerative colitis, in comparison with that in normal sera. Furthermore, the main peak of this acidic protein in Crohn's disease was an isoelectric point (pI) of 2.8, while the pI of that from ulcerative colitis was 3.0. These results suggest that qualitative differences of such acidic protein may serve to discriminate between the sera of Crohn's disease and ulcerative colitis.     

The therapeutic use of acetaminophen in patients with liver disease

Acetaminophen has been used safely and effectively for many years to manage pain and/or fever in patients of all ages. It is commonly recommended as first-line therapy for a variety of patients and conditions, including the elderly, children with viral illnesses, and patients with osteoarthritis, gastrointestinal conditions, bleeding disorders, cardiovascular disease, or renal disease. However, its use is often avoided in patients with chronic liver disease. The perception that acetaminophen should be avoided in such patients arose from awareness of the association between massive acetaminophen overdose and hepatotoxicity, combined with a lack of understanding of the metabolism of acetaminophen in patients with liver disease. There are various theoretical mechanisms of acetaminophen hepatotoxicity in chronic liver disease including: altered metabolism and depleted glutathione stores that would be expected to increase accumulation of the hepatotoxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI). Available studies in patients with chronic liver disease, however, have shown that although the half-life of acetaminophen may be prolonged, cytochrome P-450 activity is not increased and glutathione stores are not depleted to critical levels in those taking recommended doses. Furthermore, acetaminophen has been studied in a variety of liver diseases without evidence of increased risk of hepatotoxicity at currently recommended doses. Therefore, acetaminophen can be used safely in patients with liver disease and is a preferred analgesic/antipyretic because of the absence of the platelet impairment, gastrointestinal toxicity, and nephrotoxicity associated with nonsteroidal antiinflammatory drugs.     

Comparison of common gamma-chain cytokines, interleukin-2, interleukin-7, and interleukin-15 for the in vitro generation of human tumor-reactive T lymphocytes for adoptive cell transfer therapy

The adoptive transfer of human tumor-reactive T lymphocytes into autologous patients can mediate the regression of metastatic melanoma. Here, the in vitro generation of melanoma-reactive T lymphocytes was compared using 3 common gamma-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. The proliferation, function, and phenotype were evaluated for tumor-reactive T cells derived from peripheral blood mononuclear cells (PBMCs) from patients previously immunized with the melanoma-associated peptide gp100:209-217(210M) and PBMCs transduced with a retrovirus encoding the alpha and beta chains of a gp100-reactive T-cell receptor (TCR). IL-7 alone did not induce significant proliferation of any tumor-reactive T-cell population, whereas IL-2 and IL-15 induced significant proliferation of tumor-reactive T lymphocytes from both sources. Cells cultured in the presence of IL-2 or IL-15 secreted comparable amounts of interferon-gamma and IL-2 in response to melanoma cells in vitro and were phenotypically similar in terms of costimulatory molecules (CD27 and CD28), cytokine receptors (CD25, CD122, and CD127), and a lymphoid homing molecule (CD62L). In addition, the proliferation, function, and phenotype of T cells cultured with combinations of IL-2, IL-7, and IL-15 were similar to those grown with IL-2 alone. The effects of these cytokines on TCR stimulation of CD45RA+ naive cells derived from adult patients and from human umbilical cord blood were also compared. Similar to the data with activated tumor-reactive T lymphocytes, IL-7 alone did not support significant proliferation of naive T cells after TCR stimulation with anti-CD3, although IL-2 and IL-15 induced comparable proliferation of T lymphocytes with similar phenotypic attributes.     

EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2

Symptomatic primary Epstein-Barr virus (EBV) infection and elevated humoral immune responses to EBV are associated with an increased risk of developing multiple sclerosis (MS). We explored mechanisms leading to this change in EBV-specific immunity in untreated patients with MS and healthy virus carriers matched for MS-associated HLA alleles. MS patients showed selective increase of T cell responses to the EBV nuclear antigen 1 (EBNA1), the most consistently recognized EBV-derived CD4⁺ T cell antigen in healthy virus carriers, but not to other EBV-encoded proteins. In contrast, influenza and human cytomegalovirus–specific immune control was unchanged in MS. The enhanced response to EBNA1 was mediated by an expanded reservoir of EBNA1-specific central memory CD4⁺ T helper 1 (Th1) precursors and Th1 (but not Th17) polarized effector memory cells. In addition, EBNA1-specific T cells recognized myelin antigens more frequently than other autoantigens that are not associated with MS. Myelin cross-reactive T cells produced IFN-γ, but differed from EBNA1-monospecific cells in their capability to produce interleukin-2, indicative of a polyfunctional phenotype as found in controlled chronic viral infections. Our data support the concept that clonally expanded EBNA1-specific CD4⁺ T cells potentially contribute to the development of MS by cross-recognition of myelin antigens.     

Diagnosis and characterization of presymptomatic patients with Wilson's disease and the use of molecular genetics to aid in the diagnosis

Wilson's disease (WD) is an autosomal recessive disorder of copper accumulation leading to liver and/or brain damage. Although fatal if untreated, the condition can be treated effectively. Autosomal recessive inheritance indicates that siblings of affected patients are at 25% risk of having the disease. If they are diagnosed prior to becoming symptomatic, affected siblings can be kept free of symptoms by prophylactic therapy. In this paper we have examined the utility of copper-related variables, along with other clinical and molecular findings, in identifying those siblings of affected patients who should be further evaluated with a liver biopsy. Data are presented on a series of 13 presymptomatic patients in whom we have made the diagnosis of WD based on liver biopsy findings. Signs of liver disease were present in 12 out of 13 cases. The classic, noninvasive, screening approaches that we evaluated were not adequate to identify all cases of WD in this group of patients. These included positive Kayser-Fleischer (KF) rings, elevated liver serum alanine transferase, elevated urine copper, or elevated plasma nonceruloplasmin copper. We have introduced the use of molecular genetics for screening siblings of affected patients for WD. We show that a probe from the linked retinoblastoma (RB) gene can be very helpful in problem cases. However, at this time, the quantitative determination of liver copper concentration remains as the definitive diagnostic criterion.     

A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma

This report describes a phase I clinical trial using nonmyeloablative, lympho-depleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma. The chemotherapy-conditioning schedule that induced transient lymphopenia consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m(2) per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded, tumor-reactive, autologous T-cell clones selected for high avidity recognition of melanoma antigens. Cohorts of three to six patients each received either no interleukin (IL)-2, low-dose IL-2 (72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2 (720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. High dose intravenous IL-2 was better tolerated by patients after chemotherapy than during previous immunotherapy cycles without chemotherapy. No patient exhibited an objective clinical response to treatment, although five patients demonstrated mixed responses or transient shrinkage of metastatic deposits. This study established a nonmyeloablative-conditioning regimen that could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma.     

The use of physical restraints for patients suffering from dementia

This study reviews the ethical dilemmas of nursing staff about using restraints on patients suffering from dementia in two types of health care settings in Israel: internal medicine wards of three general hospitals; and psychogeriatric wards of three nursing homes. The nurses' level of knowledge about the Patient's Rights Law, the Israeli Code of Ethics, and the guidelines on restraints was analysed. The purposes of restraints were defined as beneficial to: (1) the patient; (2) other patients; or (3) the institution. The concept was evaluated in a realistic situation (expressing views of daily practice) and in an idealistic situation (expressing personal and professional beliefs and values). It was shown that nurses in internal medicine wards of general hospitals agreed more with the use of restraints than those in psychogeriatric wards in nursing homes. Differences were more pronounced when restraints were beneficial to the institution. In addition, nurses working in psychogeriatric wards of nursing homes had more knowledge about the guidelines on restraints and were less inclined than their counterparts to agree with the use of restraints for the benefit of other patients or the institution.     

Clinical applications of the 2nd generation assay for anti-TSH receptor antibodies in Graves' disease. Evaluation in patients with negative 1st generation test.

Detection of autoantibodies to the thyrotropin receptor by radioreceptor assays is largely requested in clinical practice for the diagnosis of Graves' disease and its differentiation from diffuse thyroid autonomy. Additionally, thyrotropin receptor antibodies (TRAb) measurement during antithyroid drug treatment can be useful to evaluate the risk of relapse after discontinuation of the therapy. Nevertheless, some patients affected by Graves' disease are TRAb-negative when a 1st generation assay is used. In this study we evaluated the diagnostic performance of a newly developed 2nd generation TRAb assay in 46 patients with Graves' disease with negative 1st generation TRAb assay results. A control group of 50 Graves' disease patients with positive 1st generation TRAb assay results, 50 patients with Hashimoto's thyroiditis and 50 patients with nodular goiter were also examined. Forty one of 46 patients with Graves' disease with negative 1st generation TRAb assay results showed a positive 2nd generation test. No differences were seen in control groups. In conclusion, the 2nd generation TRAb assay is more sensitive than the 1st generation test and should be used in clinical practice. Long-term prospective studies are needed to evaluate the prognostic role of the 2nd generation TRAb assay in Graves' disease.