Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation.

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.

Small molecule antibody targeting HLA class I inhibits myeloma cancer stem cells by repressing pluripotency-associated transcription factors.

Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of ß-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy.

Initial steroid hormone dose in the treatment of idiopathic sudden deafness.

OBJECTIVE: The purpose of this study was to clarify whether higher doses of steroids improve the prognosis of idiopathic sensorineural hearing loss (ISHL) and the suitable dose of steroid hormone. STUDY DESIGN: The study was a retrospective statistical analysis. SETTING: This study was performed at the Department of Otolaryngology, Head Neck Surgery, Kumamoto University School of Medicine. PATIENTS: Two hundred fifty patients with ISHL were analyzed in this study. They were divided into two groups: those receiving less than a specified daily dose of steroid and those receiving a daily dose greater than or equal to the specified dose. INTERVENTIONS: The patients received systemic steroid therapy combined with adenosine triphosphate, vitamins, diuretics, vasodilators, hyperbaric oxygen therapy, stellate ganglion block, or volume expander. MAIN OUTCOME MEASURES: The correlation between the initial dose of steroid hormone and the improvement rate was analyzed. RESULT: Spearman's correlation coefficients and partial correlation coefficients between the initial dose and the prognosis were all significantly negative. On the other hand, the correlations between the initial dose and the prognosis were positive in the group receiving <30 mg/day, whereas they were negative in the group receiving > or =30 mg/day, although these correlations were not significant. CONCLUSION: The general use of steroid hormone to treat ISHL is not recommended. Furthermore, if steroid hormone is used for treatment, the use of <30 mg/day of prednisolone is preferable.

Bone marrow-derived cells are responsible for the development of autoimmune arthritis in human T cell leukemia virus type I-transgenic mice and those of normal mice can suppress the disease.

Previously, we reported that human T cell leukemia virus type I env-pX region-introduced transgenic (pX-Tg) mice developed an inflammatory polyarthropathy associated with a development of autoimmunity. To elucidate roles of autoimmunity in the development of arthritis, the immune cells were reciprocally replaced between pX-Tg mice and non-transgenic (Tg) mice. When bone marrow (BM) cells and spleen cells from pX-Tg mice were transferred into irradiated non-Tg mice, arthritis developed in these mice. In contrast, arthritis in pX-Tg mice was completely suppressed by non-Tg BM and spleen cells. Similar results were obtained with BM cells only. After the transplantation, T cells, B cells, and macrophages were replaced completely, whereas cells in the joints were replaced partially. In those mice, serum Ig and rheumatoid factor levels correlated with the disease development, and inflammatory cytokine expression was elevated in the arthritic joints. Furthermore, involvement of T cells in the joint lesion was suggested, because the incidence was greatly reduced in athymic nu/nu mice although small proportion of the mice still developed arthritis. These observations suggest that BM stem cells are abnormal, causing autoimmunity in pX-Tg mice, and this autoimmunity plays an important, but not absolute, role in the development of arthritis in this Tg mouse.

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice.

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.

[The effect of drug therapy and stellate ganglion block with or without oxygen inhalation on sudden hearing loss].

Forty-one patients suffering from sudden hearing loss were studied by the following method. Twenty patients (group A) were treated with oral administration of prednisolone, intravenous administration of vitamin B and C, furosemide and stellate ganglion block. Another 21 patients (group B) were treated with oral administration of these drugs, stellate ganglion block and oxygen inhalation. Forty six percent of all these patients, 35 percent of group A and 57 percent of group B, regained less than 20 dB of their normal hearing level. The patients who are younger, having shorter duration from first finding of symptoms to starting of therapy and smaller average deficiency of hearing, without dizziness are easy to recover. Oxygen inhalation with drug therapy and stellate ganglion block is a useful treatment for sudden hearing loss.

Effects of ceruletide on clinical symptoms and EEGS in schizophrenia.

1. The efficacy of ceruletide as a supplement in treating schizophrenics was tested by monitoring the Brief Psychiatric Rating Scale (BPRS) and the EEG. 2. Eight male inpatients with schizophrenia were administered fixed doses of neuroleptics during the study. 3. A control EEG recording and BPRS scoring were done before ceruletide administration. 4. Doses of 0.8 micrograms/kg/week of ceruletide and of placebo were given intramuscularly in a double-blind, crossover design for 3 consecutive weeks, and no treatment followed for 1 week. 5. EEG recordings and BPRS scoring were carried out once weekly. There were no significant differences in the total BPRS scores or the scores of each item between ceruletide and placebo. 6. With ceruletide treatment, the power values of the frontal EEGs increased in the whole bands but only in the first week. 7. The EEG values in the occipital area increased in alpha and beta activities slightly in the third week and markedly in the fourth week. 8. The power values in the right temporal area decreased in fast beta activity in the second and third weeks but increased in alpha activity in the third and fourth week. 9. The power of the left temporal area increased in both alpha and beta bands in the second week, and this continued to the fourth week. 10. These results suggest that treatment with ceruletide might fail to improve the symptoms of schizophrenics but does affect their EEGs, and that ceruletide may have a delayed effect.