Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2,α was 3.4±3.1 h andt1/2,β 66.6±27.4 h in 9/10 children. Thet1/2,β values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2,β decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2

Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8–20 h/day; 4–5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5–6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.     

Tolerability,response and outcome of high-risk neuroblastoma patients treated with long-term infusion of anti-GD2 antibody ch14.18/CHO

Immunotherapy with short term infusion (STI) of monoclonal anti-GD₂ antibody (mAb) ch14.18 (4 × 25 mg/m²/d; 8–20 h) in combination with cytokines and 13-cis retinoic acid (RA) prolonged survival in high-risk neuroblastoma (NB) patients. Here, we investigated long-term infusion (LTI) of ch14.18 produced in Chinese hamster ovary cells (ch14.18/CHO; 10 × 10 mg/m²; 24 h) in combination with subcutaneous (s.c.) interleukin-2 (IL-2) in a single center program and report clinical response, toxicity and survival. Fifty-three high-risk NB patients received up to 6 cycles of 100 mg/m² ch14.18/CHO (d8–17) as LTI combined with 6 × 10⁶ IU/m² s.c. IL-2 (d1–5; 8–12) and 160 mg/m² oral RA (d19–32). Pain toxicity was documented with validated pain scores and intravenous (i.v.) morphine usage. Response was assessed in 37/53 evaluable patients following International Neuroblastoma Risk Group criteria. Progression-free (PFS) and overall survival (OS) was analyzed by the Kaplan-Meier method and compared to a matched historical control group from the database of AIEOP, the “Italian Pediatric Ematology and Oncology Association”. LTI of ch14.18/CHO showed acceptable toxicity profile indicated by low pain scores, reduced i.v. morphine usage and low frequency of Grade ≥3 adverse events that allowed outpatient treatment. We observed a best response rate of 40.5% (15/37; 5 CR, 10 PR), 4-year (4 y) PFS of 33.1% (observation 0.1- 4.9 y, mean: 2.2 y) and a 4 y OS of 47.7% (observation 0.27 – 5.20 y, mean: 3.6 y). Survival of the entire cohort (53/53) and the relapsed patients (29/53) was significantly improved compared to historical controls. LTI of ch14.18/CHO thus shows an acceptable toxicity profile, objective clinical responses and a strong signal of clinical efficacy in NB patients.     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2, was 3.4±3.1 h andt1/2, 66.6±27.4 h in 9/10 children. Thet1/2, values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2, decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439, and in part by a grant from the General Clinical Research Center Program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M.M.U.-F. and C.S.H. were in part supported by a grant from the Children's Cancer Research Foundation, and M.M.U.-F. was in part supported by a grant of the Kommission für Forschung und wissenschaftlichen Nachwuchs, Charité 95-010/9610766     

Functional Bioassays for Immune Monitoring of High-Risk Neuroblastoma Patients Treated with ch14.18/CHO Anti-GD2 Antibody

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD₂ antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD₂-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20∶1 for PBMC preparations as best suited for GD₂-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40∶1. Optimal results for CDC were found with a serum dilution at 1∶8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m²) revealed GD₂-specific increases in CDC (4.5–9.4 fold) and ADCC (4.6–6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1

Background

Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.

Methods

Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.

Results

CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.

Conclusion

Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1.     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Superantigen-staphylococal-enterotoxin-A-dependent and antibody-targeted lysis of GD2-positive neuroblastoma cells

 Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) Vβ expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD₂ human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD₂ is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch14.18 to SEA was achieved either with a protein-A–SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD₂-positive neuroblastoma cells in an effector-to-target(E:T)-ratio-and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A–SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A–SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy. Received: 15 March 1995 / Accepted: 22 May 1995     

Phagocytosis Deficiency of Macrophages in NOD.H-2h4 Mice Accelerates the Severity of Iodine-Induced Autoimmune Thyroiditis

Apoptosis occurs in many autoimmune diseases. Excess iodine induces thyrocyte apoptosis and increases the incidence and prevalence of autoimmune thyroiditis (AIT). However, the sequence of events between the appearance of thyrocyte apoptosis and the occurrence of thyroiditis remains uncharacterized. Furthermore, few studies have investigated the role of macrophage phagocytosis in the development of AIT. Therefore, we evaluated the relationship between apoptosis and inflammatory infiltration in NOD.H-2^h4 mouse thyroids by comparing the sequence of events in tissue samples. We also investigated the role of macrophages by comparing macrophage phagocytosis function in BALB/c, C57BL/6, and NOD.H-2^h4 mice treated with different levels of iodine. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and thyroid inflammatory scores revealed that apoptosis (2 weeks) occurred before inflammatory infiltration (4 weeks). Phosphatidylserine (PS) expression on the extracellular surface of the cell membrane and double-stranded DNA fragments associated with apoptosis appeared at 2 and 8 weeks, respectively. Additionally, although apoptosis was enhanced in the thyroids of mice supplemented with excess iodine (0.05 ± 0.12 vs 1.63 ± 0.82% for BALB/c, 0.09 ± 0.14 vs 1.51 ± 0.34% for C57BL/6, and 0.07 ± 1.11 vs 4.72 ± 0.62% for NOD.H-2^h4 mice), only NOD.H-2^h4 mouse thyroids presented with inflammation. Furthermore, macrophages from NOD.H-2^h4 mice (44.46 ± 1.79%) exhibited decreased phagocytotic activity relative to that in BALB/c (54.21 ± 4.58%) and C57BL/6 (58.96 ± 4.04%) mice. There were no differences in phagocytosis function between NOD.H-2^h4 mice supplemented with excess iodine or left untreated (24.50 ± 2.66 vs 21.71 ± 1.79%, p = 0.06). In conclusion, deficiencies in the apoptosis clearance of macrophages in NOD.H-2^h4 mice may constitute an early pathogenic mechanism in AIT that is not influenced by iodine intake.

     

Potential therapeutic radiotracers: preparation, biodistribution and metabolic characteristics of 177Lu-labeled cyclic RGDfK dimer

In this study, we reported the preparation and evaluation of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)]₂) as a potential therapeutic radiotracers for the treatment of integrin α_vβ₃-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100°C and higher DOTA-conjugate concentration were needed for successful ¹⁷⁷Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of ¹⁷⁷Lu-labeled cyclic RGDfK peptides, and ¹⁷⁷Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin α_vβ₃-positive tumors.     

Intratumoral treatment of smaller mouse neuroblastoma tumors with a recombinant protein consisting of IL-2 linked to the Hu14.18 antibody increases intratumoral CD8+ T and NK cells and improves survival

Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV- versus IT-treated animals. Studies presented here describe the analyses of tumor burden at the initiation of treatment and its effects on treatment efficacy, survival, and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+ T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.     

Fluctuation analyses of spontaneous mutations to 6-thioguanine resistance in Chinese hamster ovary cells in culture

Fluctuation analyses of the spontaneous appearance of 6-thioguanine (TG)-resistant mutants in cultured Chinese hamster ovary (CHO) cells were performed to investigate (1) whether the resistance is induced by the selective agent or is the result of a mutation which occurs prior to the TG selection and (2) to estimate the spontaneous mutation rate at the hypoxanthine—guanine phosphoribosyl transferase (hgprt) locus. The potential problem of phenotypic delay was minimized by allowing an adequate expression time through maintenance of the cultures in a division-arrested, viable state. The results demonstrate that the TG-resistant (TG^r) cells arise randomly in the cultures, independently of the selective agent, which is consistent with spontaneous mutations. The average values for mutation rate ± standard deviation, based on 4 independent determinations and 2 methods of calculation, are 3.4 ± 1.2 × 10^?7 (median method) and 5.1 ± 1.8 × 10^?7 (mean method) mutants/cell/generation.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis

Background

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

Methods

Immortalised fibroblasts from DBA/1 mice (DBA/1/0 cells) were transfected with a plasmid expressing IL‐4 cDNA (DBA/1/IL‐4 cells). Xenogeneic fibroblasts from Chinese hamster ovary (CHO) transfected with a plasmid expressing IL‐4 cDNA (CHO/IL‐4) were studied also. The cells were engrafted in mice developing CIA by subcutaneous injection of 3 × 10⁶ DBA/1/0 or DBA/1/IL‐4 or CHO/IL‐4 cells.

Results

Injection of DBA/1/IL‐4 cells, on days 10 and 25 after immunisation, was associated with a significant and lasting improvement in the clinical and histological evidence of joint inflammation and destruction as compared with DBA/1/0 and CHO/IL‐4 cells. DBA/1/IL‐4 cell treatment decreased also the production of IgG2a antibody to CII and the proliferation of CIIB‐specific nodal T cells. Later treatments (engraftments on days 23 and 35 after immunisation) exerted also an anti‐inflammatory effect, as evaluated on clinical and histological signs of CIA.

Conclusions

Taken together, these findings indicate that systemic administration of syngeneic cells transfected with an anti‐inflammatory cytokine gene, namely IL‐4, with a non‐viral method is effective in CIA and may attenuate the cytokine imbalance seen in this disease. Copyright © 2002 John Wiley & Sons, Ltd.

     

Equilibrium constants under physiological conditions for the condensation of acetaldehyde with tetrahydrofolic acid

The nonenzymatic reaction of ethanol-derived CH₃CHO with tissue constituents continues to be of interest as a potential mechanism underlying the toxicity of alcohol. The current study has focused on the spontaneous condensation of CH₃CHO with H₄folate under physiological conditions (38 °C, pH 7.0, I = 0.25 M). Computer analysis of uv spectral changes with increasing CH₃CHO concentrations demonstrated the presence of at least two different adducts. The observed equilibrium constant (K_obs) for the formation of the first adduct is 91 ± 2 m^?1 (121 ± 2 m^?1 at 25 °C), a value which is unaffected by variations in ionic strength (0.06–1.0 m) or by free [Mg²⁺] up to 5 mm. The NMR spectrum is compatible with the structure: 5,10-CH₃CH-H₄folate analogous to the naturally occurring 5,10-CH₂-H₄folate. The formation of the latter compound from HCHO and H₄folate, however, is much more favorable under the same conditions [K_obs = 3.0 ± 0.2 × 10⁴ M^?1 (38 °C), 3.6 ± 0.1 × 10⁴ M^?1 (25 °C)]. At the levels of CH₃CHO which accumulate during ethanol metabolism in vivo only a small fraction of the H₄folate will exist as the CH₃CHO derivative, yet it may ultimately be the ratio of free CH₃CHO to free HCHO in tissue which determines the physiological importance of the CH₃CHO adduct. Other adduct(s) of CH₃CHO with H₄folate are observed at very high levels of CH₃CHO but are unlikely to be of physiological significance.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

β‐Cell Function and Insulin Sensitivity in Adolescents From an OGTT

Given the increase in the incidence of insulin resistance, obesity, and type 2 diabetes in children and adolescents, it would be of paramount importance to assess quantitative indices of insulin secretion and action during a physiological perturbation, such as a meal or an oral glucose‐tolerance test (OGTT). A minimal model method is proposed to measure quantitative indices of insulin secretion and action in adolescents from an oral test. A 7 h, 21‐sample OGTT was performed in 11 adolescents. The C‐peptide minimal model was identified on C‐peptide and glucose data to quantify indices of β‐cell function: static φ_s and dynamic φ_d responsivity to glucose from which total responsivity φ was also measured. The glucose minimal model was identified on glucose and insulin data to estimate insulin sensitivity, S_I, which was compared to a reference measure, S_I^ref, provided by a tracer method. Disposition indices, which adjust insulin secretion for insulin action, were then calculated. Indices of β‐cell function were φ_s = 51.35 ± 8.89 × 10^?9min^?1, φ_d = 1,392 ± 258 × 10^?9, and φ = 82.09 ± 17.70 × 10^?9min^?1. Insulin sensitivity was S_I = 14.19 ± 2.73 × 10^?4, not significantly different from S_I^ref = 14.96 ± 3.04 × 10^?4 dl/kg·min per µU/ml, and well correlated: r = 0.98, P < 0.0001, thus indicating that S_I can be accurately measured from an oral test. Disposition indices were DI_s = 1,040 ± 201 × 10^?14 dl/kg/min² per pmol/l, DI_d = 33,178 ± 10,720 × 10^?14 dl/kg/min per pmol/l, DI = 1,844 ± 522 × 10^?14 dl/kg/min² per pmol/l. Virtually the same minimal model assessment was obtained with a reduced 3 h, 9‐sample protocol. OGTT interpreted with C‐peptide and glucose minimal model has the potential to provide novel insight regarding the regulation of glucose metabolism in adolescents, and to evaluate the effect of obesity and interventions such as diet and exercise.     

Effects of Alleles at the W Locus on Testicular Luteinizing Hormone Receptors in Adult Mice

Abstract

The autoregulation of testicular LH receptors was studied in W^x/W^v mice with germ cell aplasia and in normal (±/±) mice. To assess the effects of each individual allele, W^x/± and W^v/± mice were also examined. Basal testicular LH receptor concentration was higher in W^x/W^v mice than in all other genotypes, and higher in W^x/± than in ±/± mice. Twenty-four h after injection of 0.3 IU hCG/g bw, LH receptor concentration was decreased in ±/± and W^v/± mice, but not in W^x/W^v or W^x/± animals. Administration of hCG caused a significant increase in plasma testosterone levels in all genotypes. However, injection of the highest dose of hCG used (0.9 IU/g bw) caused a significantly greater elevation in plasma testosterone in W^x/W^v than in ±/± mice. Plasma gonadotropin levels were significantly higher in W^x/W^v mice than in all other genotypes. The present results indicate that the W^x allele is responsible for the changes in testicular function observed in W^x/W^v mice, and suggest that this allele may be involved in the genetic regulation of testicular LH receptors in the mouse.