Interleukin 10 secretion and impaired effector function of major histocompatibility complex class II-restricted T cells anergized in vivo.

Continuous antigenic stimulation in vivo can result in the generation of so-called "anergic" CD4(+) or CD8(+) T cells that fail to proliferate upon antigenic stimulation and fail to develop cytolytic effector functions. Here we show that class II major histocompatibility complex-restricted T cells specific for influenza hemagglutinin (HA) that become anergic in mice expressing HA under control of the immunoglobulin kappa promoter exhibit an impaired effector function in causing diabetes in vivo, as compared to their naive counterparts, when transferred into immunodeficient recipients expressing HA under the control of the insulin promoter. Furthermore, HA-specific T cells anergized in vivo contain higher levels of interleukin (IL)-4 messenger RNA (mRNA) than naive and recently activated T cells with the same specificity and more than a 100-fold higher levels of IL-10 mRNA. The higher expression of the IL-10 gene is also evident at the protein level. These findings raise the interesting possibility that T cells rendered anergic in vivo have in fact become regulatory T cells that may influence neighboring immune responses through the release of IL-10.

Allogeneic stem cell transplantation from related and unrelated donors for aplastic anaemia in adults--a single-centre experience.

Aplastic anaemia (AA) is a rare bone marrow failure syndrome treated either by immunosuppressive therapy or allogeneic stem cell transplantation (SCT). At present, no randomised clinical trials evaluating both treatment options, and in particular SCT from unrelated donors, are available. We here report the clinical course and outcome of allogeneic SCT for 20 consecutive adult patients with AA. Newly diagnosed and untreated patients (n = 8) or patients pre-treated by immunosuppressive therapy (n = 12) were transplanted either from human-leukocyte-antigen (HLA) identical family donors (n = 13) or matched (n = 6) and mismatched (n = 1) unrelated donors, respectively. Conditioning varied depending on donor type and included cyclophosphamide with or without anti-thymocyte globulin (ATG) and fludarabine-cyclophosphamide-ATG with or without low-dose total body irradiation. With a median follow-up of more than 40 months, all patients have had favourable outcomes with stable haematopoietic engraftment and high performance scores. Six patients developed acute (five I degrees -II degrees ; one >II degrees ) and four limited chronic graft-versus-host disease. In this group of AA patients, allogeneic SCT has proven very successful, independent of donor type and pre-treatment. Studies with greater cohorts of patients are warranted to better determine indication and timing of SCT especially from unrelated donors in AA.

Fingerprints of anergic T cells.

Peripheral T cell tolerance may result from activation-induced cell death [1], anergy [1], and/or immune response modulation by regulatory T cells [2]. In mice that express a transgenic receptor specific for peptide 111-119 of influenza hemagglutinin presented by E(d) class II MHC molecules as well as hemagglutinin under control of the immunoglobulin-kappa promoter, we have found that anergic T cells [3] can also have immunoregulatory function and secrete IL-10 [4]. In order to obtain information on molecular mechanisms involved in anergy and immunoregulation, we have compared expression levels of 1176 genes in anergic, naive, and recently activated CD4+ T cells of the same specificity by gene array analysis. The results provide a plausible explanation for the anergic phenotype in terms of proliferation, provide new information on the surface phenotype of in vivo-generated anergic CD4+ T cells, and yield clues with regard to new candidate genes that may be responsible for the restricted cytokine production of in vivo-anergized CD4+ T cells. The molecular fingerprints of such T cells should enable the tracking of this small population in the normal organism and the study of their role in immunoregulation.

Migratory properties of ex vivo expanded regulatory T cells: Influence of all-trans retinoic acid and rapamycin.

Adoptively transferred regulatory T-cells represent a promising therapeutic approach for tolerance induction in autoimmunity and transplantation medicine. However, a major hurdle for clinical application is the manufacturing of sufficient Treg cell numbers with respect to the low frequency of naturally occurring Tregs in the peripheral blood. Therefore, ex vivo large-scale expansion is mandatory for most of the clinical conditions. Besides the Treg cell number other parameters of the cell product are of high relevance for safe and efficient clinical Treg cell application like Treg cell purity, suppressive capacity and genetic stability of the Treg cell phenotype. Moreover, migratory properties of ex vivo expanded Tregs should be defined very clearly in order to predict their migration to secondary lymphoid organs as sites of antigen-specific activation, in vivo proliferation and subsequent trafficking to affected target organs. Therefore, we studied different cell culture conditions for Treg large-cell expansion using all-trans retinoic acid (ATRA) and/or rapamycin (Rapa) with focus on their migratory properties. The tested culture conditions revealed comparable chemokine receptor expression profiles (CXCR3, CCR4, CCR6, CCR7) and functional migration capabilities (IP10 and CCL19) with respect to Th1 and Th2 inflammatory conditions. However, the most striking difference was detected for the expansion capacity, suppressive potency and genetic stability likely predisposing large-scale expansion with ATRA and/or Rapa for therapeutic intervention in acute GvHD and without supplementation for chronic GvHD.

Autoimmunity resulting from cytokine treatment predicts long-term survival in patients with metastatic renal cell cancer.

PURPOSE: In patients undergoing cytokine therapy, systemically applied interleukin-2 (IL-2) and/or interferon-alpha (IFN-alpha) have been reported to induce thyroid dysfunction as well as thyroid autoantibodies. We analyzed the correlation of thyroid autoimmunity with HLA phenotype, various other autoimmune parameters, and patient survival. PATIENTS AND METHODS: For this purpose, antithyroglobulin autoantibodies, antimicrosomal thyroid autoantibodies, thyroglobulin receptor autoantibodies, thyroid dysfunction, and multiple clinical parameters were determined in 329 unselected patients with metastatic renal cell cancer before and after systemic IL-2 and IFN-alpha2 therapy. For statistical analysis, we used both univariate and multivariate Cox proportional hazards models and the two-tailed Fisher's exact test. RESULTS: Antithyroglobulin autoantibodies and antimicrosomal thyroid autoantibodies were detected in 60 patients (18%); positive autoantibody titers of various other autoimmune parameters were statistically unrelated. The presence of thyroid autoantibodies was correlated with prolonged survival (P<.0001). There was a statistically significant difference in frequencies of HLA-Cw7 expression between thyroid autoantibody-positive and -negative patients (P< or =.05), and the Cw7 expression was associated with prolonged overall survival (P = .009). CONCLUSION: The evaluation of thyroid autoantibodies during cytokine therapy could be a useful prognostic marker for patients with renal cell carcinoma who benefit from cytokine treatment. IL-2- and IFN-alpha2-induced tumor control and prolonged survival may require breaking of immunologic tolerance against self-antigens.

Chemoimmunotherapy of advanced malignant melanoma: sequential administration of subcutaneous interleukin-2 and interferon-alpha after intravenous dacarbazine and carboplatin or intravenous dacarbazine, cisplatin, carmustine and tamoxifen.

Both chemotherapy and interleukin-2 and/or interferon-alpha produce objective responses in a proportion of advanced malignant melanoma patients. While duration of response to chemotherapy is short, i.e. usually below 4 months, immunotherapy has resulted in a small number of long-lasting remissions in patients with metastatic melanoma. In two consecutive phase II trials in a total of 67 patients, we assessed the potential synergism between both modalities, i.e. chemo- and immunotherapy. Treatment consisted of intravenous (i.v.) carboplatin (CBDCA, 400 mg/m2) and dacarbazine (DTIC, 750 mg/m2) given twice (i.v. bolus over 30 min) at 3-week intervals, or 4 cycles of DTIC (220 mg/m2 i.v. 3 days), cisplatin (DDP, 35 mg/m2 i.v. 3 days), carmustine (BCNU, 150 mg/m2 i.v. cycles 1 and 3) and tamoxifen (TAM, 20 mg oral/daily) at 3-week intervals. Chemotherapy was followed by immunotherapy with combined subcutaneous (s.c.) interleukin-2 (rIL-2) and SC interferon-alpha 2 (rIFN-alpha). Among 40 patients who received a full cycle of chemotherapy with CBDCA/DTIC and sequential immunotherapy, there were 3 (7.5%) complete remissions (CRs) with a median duration of 19 months (range 13-26+). Partial remissions (PRs) were noted in 11 (27.5%) patients with a median response duration of 8 (range 5-14) months. Among 27 patients who received DTIC/DDP/BCNU/TAM and rIL-2/rIFN-alpha, there were 3 (11%) complete remissions and 12 (44.5%) partial remissions. Duration of complete and partial remissions ranged from 9+ to 13+ (median, 11+), and 5 to 15+ (median, 7+) months, respectively. Chemotherapy produced mostly moderate toxicity. Thrombocytopenia was common with the nadir after a median time of 18 days following start of CBDCA/DTIC and DTIC/DDP/BCNU, respectively. 10 patients required transfusion of thrombocytes. Nausea and vomiting due to chemotherapy were well tolerated using concomitant ondansetrone (8 mg i.v.). Immunotherapy was self-administered at home with mild to moderate side effects; malaise, fever, chills, nausea/vomiting, diarrhoea, anorexia and arthralgias were most frequent, but were spontaneously reversible after ending rIL-2/IFN-alpha. A mean 87 and 88% of the projected doses of rIL-2 and rIFN-alpha were administered on either protocol. There were no life-threatening complications and no treatment-related deaths. The sequential combination of chemotherapy and rIL-2 plus rIFN-alpha had at least additive therapeutic activity against metastatic malignant melanoma. The schedules produced long-lasting remissions and were tolerated well overall. These trials substantiate a potential role for low to intermediate dose immunotherapy in maintaining and consolidating therapeutic effects of chemotherapy in metastatic melanoma.

Molecular and prognostic classification of advanced melanoma: a multi-marker microcontamination assay of peripheral blood stem cells.

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

Tumour microdissemination and survival in metastatic melanoma.

The value of tyrosinase messenger RNA (mRNA) detection by reverse-transcriptase polymerase chain reaction (RT-PCR) as a marker for circulating melanoma cells remains controversial. However, it has been suggested that detection of melanoma cell mRNA may be used to evaluate prognosis and disease progression in patients with advanced malignant melanoma. We used a highly sensitive tyrosinase RT-PCR detection assay to test peripheral blood specimens of 80 patients with metastatic malignant melanoma. Moreover, we developed a multiple marker RT-PCR assay detecting a variety of additional melanocyte/tumour specific markers addressing the potential heterogeneity of gene expression of circulating melanoma cells. Thus subgroups of 32 and 12 out of all the 80 patients were also analysed for multimarker gene expression in their peripheral blood and bone marrow specimens, respectively. Altogether, 15 out of 80 patients tested positive for one or more molecular markers with heterogeneous melanocyte/tumour gene expression patterns. All RT-PCR positive patients presented with progressive and widely disseminated disease. We concluded that the detection of melanoma cell mRNA occurs in a stage of massive tumour progression and may predict poor clinical outcome in advanced malignant melanoma patients (p < 0.001). In addition, the multiple marker RT-PCR analysis was more reliable and sensitive than a single molecular marker assay for the detection of melanoma cells.

Comparative study of the acute nephrotoxicity from standard dose cisplatin +/- ifosfamide and high-dose chemotherapy with carboplatin and ifosfamide.

The nephrotoxic effects of different platinum compounds based combination chemotherapies were compared. Chemotherapy consisted of either cisplatin fractionated over 5 days (5 x 20 mg/m2) or given as a single-day infusion (1 x 50 mg/m2) plus ifosfamide (4 g/m2) or high-dose chemotherapy was applied including carboplatin (3 x 500 mg/m2) and ifosfamide (3 x 4 g/m2) fractionated over three consecutive days. Conventional parameters such as serum creatinine and glomerular filtration rate (GFR), as well as urinary protein excretion of N-acetyl-beta-D-glucosaminidase (NAG)) and alpha 1-micro-globulin were assessed in 52 patients. Fractionation over 5 days without adding other nephrotoxic agents, i.e. ifosfamide, prevented decreases in GFR following cisplatin, whereas the combination of conventional dose cisplatin and ifosfamide, given as a single-day infusion, and high-dose carboplatin/ifosfamide yielded a pronounced fall of GFR. All groups showed increases in the urinary excretion levels of serum derived proteins and NAG, but with significant differences; about 2 to 3-fold for 5-days cisplatin, 3 to 5-fold for single-day cisplatin/ifosfamide, and 20 to 35-fold for high-dose chemotherapy. Thus, conventional approaches can reduce but not prevent the nephrotoxicity of cisplatin-based chemotherapy. In particular, high-dose chemotherapy regimens including carboplatin and ifosfamide are associated with comparable or even higher nephrotoxicity to single-day cisplatin/ifosfamide. In the light of the long-term consequences of persistent renal damage prevention of nephrotoxicity should be further improved.

Clinical and in vitro response to 13-cis-retinoic acid in interferon-alpha resistant renal cell carcinoma.

Retinoids are known to control many important biological processes, including differentiation, morphogenesis, growth and tissue homeostasis. More recently, clinical and pre-clinical results provide evidence for an antiproliferative effect of 13-cis-retinoic acid (13cRA) in interferon-alpha (IFN-alpha) treated renal cell carcinoma patients. The manner in which 13cRA augments antitumor effects and modulates biologic and clinical responses of renal cell carcinoma to IFN-alpha remains elusive. In the present study, we report induction of apoptosis and objective tumor regression in response to 13cRA in advanced renal cell carcinoma patients refractory to IFN-alpha. Among 21 patients treated there were one complete and four partial remissions (objective response rate, 24%; median response duration 8+ months). Preliminary evidence suggests that 13cRA acid may reverse IFN-alpha resistance in renal cell carcinoma.

Linomide and interleukin-2 in patients with advanced renal cell carcinoma.

Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic and non-immunologic mechanisms. It has shown efficacy against various malignancies, experimental autoimmune encephalomyelitis, and septic shock in animal models and has been investigated for clinical use in minimal residual myeloid leukemia with promising results. Interleukin-2 has shown considerable efficacy in palliative anti-tumor-treatment of advanced renal cell cancer, revealing remission rates of up to 40% in combination therapy regimens. Linomide is reported to exhibit synergistic effects with interleukin-2. Here we report on a clinical phase I/II study examining tolerance and efficacy of a combination therapy schedule of SQ interleukin-2 and PO Linomide in advanced renal cell cancer. Seventeen patients received 10 IU/m2 interleukin-2 per week for 8 weeks, resting interleukin-2 for another 8 weeks. In week 5 they started 5 mg Linomide daily, continued with 10 mg from week 7 to 16. No objective remissions were observed. Among 15 patients evaluable for response, 10 (66.7%) were progredient during the study. Three patients died during the observation period, including two not evaluable for response. Median survival was 4.0 months, median progression-free survival 2.5 months with a Kaplan-Meier estimate of 3.63 months. Fever, reduced general condition, nausea/vomiting, dyspnea, anorexia, chills and hypotension were the most common side effects, reaching WHO grade 3 in 6 and grade 4 in 2 cases. In summary, Linomide in combination with interleukin-2 provides no advantages in efficacy or toxicity over other therapy regimens employing interleukin-2.

HLA phenotype and cytokine-induced tumor control in advanced renal cell cancer.

BACKGROUND: The natural history of malignancies, the response to cytokine-based therapy and survival of patients may be partly determined by the human leukocyte antigen (HLA) phenotype. Here, we investigated in a retrospective analysis the correlation of the HLA phenotype of 73 prognostic favored patients with advanced renal cell carcinoma to (a) the expected HLA distribution in Caucasians, (b) the susceptibility or resistance to metastatic sites, (c) response to cytokine-based therapy and (d) sustained cytokine-induced effective tumor control. METHODS: We retrospectively determined the MHC class I and II antigens in patients with metastatic renal cell carcinoma selected by survival. Antigens were serologically typed by standard lymphocytotoxicity techniques. For statistical analysis, we calculated the probability of the presented HLA antigens in correlation to the expected Caucasian HLA phenotypes. An independent confirmation was performed by using the chi-square and two-tailed Fisher's exact test. RESULTS: Various HLA antigens deviated significantly from the normal distribution in the Caucasian population. HLA.B44 was the only antigen associated (p < 0.01) with the absence of lung and presence of bone metastases, while it did not impact on overall survival or response to therapy. A1 (p < 0.0001, p < 0.002) and B8 (p < 0.009, p < 0.04) alleles were more frequently expressed in responding patients than expected from the normal distribution in Caucasians and that observed in non-responding patients, respectively. The HLA analysis of patients achieving a durable complete remission showed a significantly higher frequency of expression of the A1 and B8 antigens and furthermore of the B14 antigen (p < 0.05). CONCLUSIONS: Our data underline the pivotal role of the MHC complex in controlling and regulating the cellular immune response in renal cell cancer. We could identify HLA antigens, which correlate with response to cytokine-treatment, with a long-lasting effective tumor control and prolonged overall survival.

Regulatory T-cells in the control of immunological diseases.

The immune system is challenged by randomly generated immune receptors that by chance can recognize self-antigens. Immunological tolerance functions as a fundamental concept in the control of a broad spectrum of immune responses not only to autoantigens but also to foreign antigens. During the past decade, CD4+ CD25+ regulatory T-cells (Tregs) have emerged as key players in the development of immunological tolerance. This review will present an update on the current knowledge about the phenotype, function, and clinical relevance of this regulatory T-cell population. The therapeutical potential of Tregs to specifically suppress immune responses in autoimmunity and transplantation and their inhibitory effects in anti-tumor immune responses will be discussed.

XYY syndrome, stigmatization, social class, and aggression: study of 15 cases.

This is a paper about the relationship of stigmatization to social class in XYY individuals, and of this relationship to manifestations of agressive behavior. The sample comprised 15 men and youths with the XYY syndrome; 8 were identified in a survey of institutions for males with behavior disorders and 7 were privately referred. All had been stigmatized by physique and appearance and/or behavior. The most frequent stigmatizing features were excessive height for age, excessive impulsiveness in behavior, and excessive temper tantrums in childhood; these were present in all cases. Antisocial or law-breaking behavior was present in 13 cases. Stigmatization did not correlate with socioeconomic class, rated according to the Shevky and Bell scale as modified to suit the study. However, the consequences of stigmatization were class related in that the lower the class, the greater the chance that the XYY individual would spend time in the penal as well as the medical system of society.

Blood dendritic cells carry terminal complement complexes on their cell surface as detected by newly developed neoepitope-specific monoclonal antibodies.

Blood dendritic cells carry the terminal complement complex (TCC) on their surface, as detected by three monoclonal antibodies (mAb). Two of these mAb were generated by immunizing mice with the terminal complement complex, whereas the third was generated by immunizing mice with blood dendritic cells. All three mAb are directed against neoepitopes on the C9 molecule, as assessed by binding and blocking experiments and studies with several forms of denatured C9 and C9-depleted serum. Only one of these mAb binds to soluble polymerized C9. All three mAb allow the quantification of human TCC in sensitive ELISA procedures and could be used as markers for the evaluation of the functions of non-lytic TCC on dendritic cells.

Molecular signals for late Tertiary/early Quaternary range splits of an Eurasian steppe plant: Clausia aprica (Brassicaceae).

Several vegetation belts stretch continuously from Europe to Asia, taiga and steppe being most prominent. Numerous plant species within these belts share a conspicuous distribution area, which is longitudinally contracted or disrupted approximately along longitude 70 degrees E. To date no hypothesis for this intriguing distribution pattern has been put forward. We detected molecular footprints in the contemporary genetic composition in nuclear DNA (ITS1, ITS2) and chloroplast DNA (trnL-trnF spacer region) of the steppe element Clausia aprica (Brassicaceae) providing evidence for a severe longitudinal range split and genetic differentiation east of the Ural Mountains about 1 million years ago caused by Quaternary climatic oscillations. Clausia aprica provides the first phylogeographical analysis on the intraspecific evolution of an Eurasian steppe plant.

Changes in function of antigen-specific lymphocytes correlating with progression towards diabetes in a transgenic model.

Mice that express influenza hemagglutinin under control of the rat insulin promoter (INS-HA) as well as a class II major histocompatibility complex (MHC)-restricted HA-specific transgenic TCR (TCR-HA), develop early insulitis with huge infiltrates, but progress late and irregularly to diabetes. Initially, in these mice, INS-HA modulates the reactivity of antigen-specific lymphocytes, such that outside the pancreas they do not cause lethal shock like their naive counterparts in single transgenic TCR-HA mice, when stimulated with high doses of antigen. Inside the pancreas, the antigen-specific cells do not initially attack the islet cells, and produce some IFN-gamma as well as IL-10 and IL-4. Spontaneous progression to diabetes, which can be accelerated by cyclophosphamide injection, is accompanied by a 10-fold increase in IFN-gamma and a 3-fold decrease in IL-10 and IL-4 production by the locally residing antigen-specific T cells. Also, total islets from non-diabetic mice contain more TNF-alpha, compared with diabetic mice. This scenario is consistent with the view that beta cell destruction depends upon the increased production of certain pro-inflammatory cytokines by infiltrating T cells. Our inability to detect Fas expression on beta cells, but not on lymphoid cells, in diabetic and non-diabetic mice, puts some constraints on the role of Fas in beta cell destruction.

Capecitabine in the treatment of metastatic renal cell carcinoma.

To evaluate the therapeutic effects and systemic toxicities of a capecitabine-based home therapy regimen in patients with metastatic renal cell carcinoma, 30 patients were enrolled in a phase II clinical trial. Treatment consisted of oral capecitabine combined with subcutaneous recombinant human interferon-alpha 2a, recombinant human interleukin-2 and oral 13-cis-retinoic acid. There were two (7%) complete responses (CRs) and eight (27%) partial remissions (PRs), for an overall objective response rate of 34% (95% CI 17-53%). Except one, all responses are ongoing, with a median duration of 9+ and 8+ months for CRs and PRs, respectively. Additionally, 12 patients (40%) reached stable disease. Eight patients (27%) showed continued disease progression despite treatment. Therapy was well tolerated and was given in the outpatient setting. Capecitabine-related World Health Organization (WHO) grade 2 and 3 toxicities were observed in five and two patients respectively, and were limited to fatigue, nausea/vomiting, diarrhoea, stomatitis, dermatitis and hand-and-foot syndrome. The substitution of capecitabine for 5-FU in the pre-existing biochemotherapy regimen did not result in a reduced therapeutic efficacy and showed significant anti-tumour activity in patients with advanced renal cell carcinoma.