Biomedical subjects
Publications and source records attributed to M Azuma.
Selenite overdose cataract, an experimental model of nuclear cataract produced in young rats is reviewed. Topics include procedures for cataract production and assessment, metabolic and molecular changes in the epithelium of the lens, calcium accumulation, activation of calcium-activated protease system, mechanisms for crystallin precipitation, anti-cataract drug testing and relevance to human cataract.
B7 molecules provide an important co-stimulatory signal for T cell receptor/CD3-mediated T cell activation via binding to their cognate receptors, CD28 and CTLA-4. We have introduced B7-1 (CD80) into M1 cells, spontaneously occurring mouse myelocytic leukemic cells, and assessed its potential to induce antitumor immunity to leukemia cells. Syngeneic, immunocompetent SL mice receiving two independent B7-1-transduced monoclonal sublines, M1-B7-1/F/clone F20 and M1-B7-1/F/clone F7, were rejected in 57% and 43% of SL mice, respectively. In vivo depletion of T cell subsets showed that both CD4+ and CD8 T cells were indispensable for the B7-1-dependent anti-leukemic immunity. Although a single exposure of irradiated monoclonal M1-B7/1/F cells was not fully effective, multiple exposures induced protective immunity against subsequent challenge with parental M1 cells. Furthermore, multiple vaccinations with irradiated monoclonal M1-B7-1/F/clone F7 cells could cure 67% of mice previously injected with a lethal number of M1 cells. These results emphasize that multiple exposures of irradiated B7-1-transduced myeloid leukemic cells can induce protective and therapeutic immunity against leukemia and that B7-1-mediated gene therapy may have therapeutic efficacy for patients with acute myelocytic leukemia.
Using 2 in vitro systems in which normal human salivary gland cells can be immortalized by transfection with SV40 DNA (NS-SV-DC), and in which a non-metastasizing human salivary gland adenocarcinoma cell clone (HSGc) can be converted to biologically aggressive and metastasizing cancer cells, we analyzed the relationship between the expression of basic fibroblast growth factor (bFGF) and malignant phenotype in human salivary gland cell clones. By enzyme immunoassay, intracellular contents of bFGF were equally detected in all the cell clones, while bFGF contained in serum-free conditioned medium of each cell clone was different: HSGc and metastasizing cell clones, respectively, released 2- and 10-fold more bFGF into media than did NS-SV-DC. Immunoblot analysis also revealed a similar result as that detected by enzyme immunoassay. By Northern blot analysis, bFGF mRNA expression was consistently detected in all the cell clones examined. We then quantitated bFGF contents in sera of tumor-bearing mice by enzyme immunoassay. Although sera from nude mice bearing an HSGc tumor showed significantly higher amounts of bFGF than those of control mice, bFGF concentrations in sera gradually decreased after removal of the tumor. For metastasizing cell clones, serum concentrations of bFGF were 2.2- to 2.6-fold higher than those of HSGc. In addition, bFGF amounts in sera after removal of the tumors were significantly higher in mice bearing metastasizing cell clone tumors compared with those of HSGc tumor-bearing mice. Our results, therefore, indicate that increase in bFGF release occurs along with the progression of human salivary gland cancer cells and that serum bFGF may be useful for evaluation of tumor presence.
In the present experiments, costimulatory effects of interleukin 12 (IL-12) and B7-2 on interleukin 2 receptor alpha chain (IL-2R alpha) expression and IL-2-dependent proliferation of naive and memory CD4+ T cells were studied in comparison with the effect of these molecules on Th1 clones. The naive and memory T cells were negatively sorted on the basis of the expression of CD44 and CD45RB, respectively, to diminish the effect of these antibodies on the activation of these cells. When these three T cell populations were stimulated with allogeneic B cells, the addition of IL-12 elicited IL-2R alpha expression and proliferation of all these T cell populations. With anti-B7-2 being included in culture, these responses of naive T cells were abrogated, whereas those of memory T cells and Th1 clones were affected only marginally. When they were stimulated with immobilized anti-CD3, the inclusion of either IL-12 or Chinese hamster ovary cells expressing B7-2 (B7-2CHO) induced IL-2R alpha expression and enhanced IL-2-dependent proliferation of memory T cells, whereas these responses of naive T cells were induced by the inclusion of B7-2CHO and enhanced by the addition of IL-12, although the addition of IL-12 alone did not affect the responses. The responses of Th1 clones were markedly enhanced by IL-12, but not by B7-2CHO, and the enhancement of the proliferation was augmented further by the addition of both IL-12 and B7-2CHO. In IFN-gamma production B7-2 costimulation was required for the enhancing effect of IL-12 on the responses of naive T cells. IL-2 production of all T cell populations was not affected by IL-12 even in the presence of B7-2CHO. These results indicate that IL-12 provides the costimulation for IL-2R alpha expression and IL-2-dependent proliferation of memory T cells and Th1 clones stimulated with TCR ligation, whereas naive T cells require B7-2 costimulation for their response to IL-12. Possible mechanisms of B7-2 costimulation in the response of T cells to IL-12 is discussed.
A vigorous production of human T lymphotropic virus type I (HTLV-I)-infected CD4+ T cells is closely associated with the development of adult T cell leukemia (ATL) and neurological disease. However, the immunological mechanisms leading to generation of the HTLV-I-infected cells are not fully clarified. The modulation of CD80 and CD86 expression on the HTLV-I-infected cells and its physiological role in the interaction of infected CD4+ T cells with uninfected CD4+ T cells was examined. The HTLV-I-infected CD4+ T cell lines established from ATL patients and normal donors by infecting their CD4+ T cells with the virus expressed CD80, CD86, and HLA-DR, and induced a proliferation of autologous and allogenic CD4+ T cells. While the CD4+ T cells stimulated with the autologous HTLV-I-infected cells for 7 days expressed CD80 and CD86 but not HTLV-I gene products, they expressed HTLV-I gag antigen after 4 weeks. The interaction of HTLV-I-infected and -uninfected CD4+ T cells was profoundly suppressed by a combination of CD80 and CD86 monoclonal antibodies. These results suggest that the induction of CD80 and CD86 on HTLV-I-infected CD4+ T cells participates actively in the generation of the virus-infected progenitor cells.
The purpose of these experiments was to develop a hypothesis to explain activation of m-calpain in cataractogenesis observed in rodents. The in vitro model used to study m-calpain activation was to correlate breakdown of the 'reporter' protein alpha-crystallin with the appearance of activated m-calpain using protein sequencing and casein zymography. Incubation of alpha-crystallins with m-calpain and Ca2+ caused proteolysis of alpha-crystallins and accumulation of new polypeptides. E64 and calpain inhibitor I each inhibited proteolysis of alpha-crystallins. The N-terminus of the 80 kDa subunit of m-calpain was blocked at time 0 (pro calpain). After incubation with Ca2+, the remaining 80 kDa subunit of m-calpain gave a N-terminal sequence of KDREAAEGLG, indicating loss of nine amino acid from the N-terminus (autolysed calpain). The new 43 kDa m-calpain fragment also gave a N-terminal sequence of KDREAAEGLG, indicating the same loss of the first nine amino acids on the N-terminus as well as a major loss of the C-terminal half of the subunit (degraded calpain). In contrast, the N-terminus of the 80 kDa subunit of m-calpain remained blocked when E64 was present (unautolysed form). Moreover, the Ca2+ concentration required for proteolysis decreased when calpain was pre-incubated with Ca2+, although proteolysis of alpha-crystallin required a higher Ca2+ concentration than proteolysis of casein. These data suggested that the sequence of events for m-calpain activation were unautolysed, autolysed and finally degraded calpain. Unautolysed and/or autolysed calpains may be proteolytically active against alpha-crystallin.
The purpose of the present investigation was to compare in vitro light scattering in the soluble proteins from rodent lenses after hydrolysis by the calcium-activated protease, m-calpain (EC 3.4.22.17). Light scattering was measured in solutions of lens proteins from mice, rats, and guinea pigs after activation of endogenous m-calpain or after addition of purified m-calpain. We found for the first time that, in addition to rat, crystallins from another rodent lens, young mouse, were susceptible to calpain-induced light scattering. As in rats, aging of mouse lens prevented calpain-induced light scattering. Although crystallins from guinea pig lens were also partially hydrolysed by calpain, appreciable light scattering did not occur. Limited proteolysis may cause common changes in the biophysical properties of mouse and rat crystallins to decrease their solubility. Discovery of the nature of these biophysical changes may help our understanding as to why crystallins precipitate under cataractous conditions.
Three non-sense mutants of varicella-zoster virus (VZV) thymidine kinase (TK) gene, VZTK325, VZTK278 and VZTK224, were isolated. The mutants had a single nucleotide substitution at codons 326, 279 and 225, which changed the codon of TGG (tryptophan) to the stop codon TGA. The wild type (WT) and mutant TKs were expressed in E. coli cells and their characteristics were evaluated. VZTK224 lost TK activity, but VZTK325 and VZTK278 maintained 74.8% and 21.2% of the WT TK activity. On the other hand, all mutants lost the thymidylate kinase activity. Moreover, VZTK325 and VZTK278 polypeptides were heat-labile. These data suggest that the carboxy-terminal portion of herpesvirus TK plays an important role in the stable folding of TK and thymidylate kinase activity.
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OBJECTIVES: This study investigated the effects of midazolam, a sedative, on tactile and pain sensations on the skin of the chin. STUDY DESIGN: Thirty-seven volunteers were segregated into four groups; the first group was the control group; the second to fourth groups were administered 0.025 mg/kg, 0.05 mg/kg, and 0.075 mg/kg of midazolam, respectively, as a bolus injection. All volunteers reclined in a dental chair for the experiment. Tactile and pain sensations were determined over time after injection of midazolam, the former using von Fray thread, the latter using an esthesiometer. RESULTS: Thresholds of tactile sensitivity and of pain were statistically significantly different from control values at 10 minutes after injection of midazolam in the 0.05 mg/kg group and in the 0.075 mg/kg group. CONCLUSION: Although 0.025 mg/kg of midazolam produced sedation, at least 0.05 mg/kg of this agent was required to alter the thresholds for perception of tactile and painful stimulation.
A patient with locally-advanced submandibular adenoid cystic carcinoma with poorly differentiated solid type, was treated with differentiation-inducing agent, vesnarinone, per os at a dose of 60 mg/day daily for 8 weeks. The vesnarinone administration caused marked regression of the tumour. In addition to conversion into the well-differentiated tubular type from the poorly differentiated solid type, the induction of apoptosis and LeY antigen was observed in the treated tumour. These findings indicate that vesnarinone might be a useful therapeutic agent for treatment of salivary cancer. Since we found the new expression of LeY antigen in the well-differentiated tubular lesion in the salivary adenoid cystic carcinoma treated with vesnarinone, we examined the LeY antigen expression in relation to tumour differentiation in five cases of salivary adenoid cystic carcinoma. Consequently, tissue sections from all of the adenoid cystic carcinoma examined showed no positive LeY staining, except for some areas in the tumour lesion with the tubular pattern including the histologically normal-appearing tissue adjacent to the tumour tissue. These findings suggest that there is the intimate relationship between the LeY antigen expression and tumour differentiation in human salivary adenoid cystic carcinoma.
Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expression of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p< 0.05) and DNA fragmentation rate (p< 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p< 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.
A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.
There are two major forms of the Fas molecule, membranous Fas and soluble Fas (sFas). To clarify the clinical significance of sFas in autoimmune diseases, we designed a sandwich ELISA to determine serum concentrations of sFas and its molecular structure, and we then analysed the correlation between levels of sFas and laboratory findings in patients with SLE and other autoimmune diseases. The levels of serum sFas were significantly higher in SLE patients than in subjects with other autoimmune diseases and in healthy donors, and the frequency of a positive serum sFas was much greater in SLE patients with high SLE disease activity index scores than in those with low scores. In addition, sFas-positive SLE patients showed a significant difference in various laboratory parameters from sFas-negative SLE patients. Serial measurements of serum sFas levels in SLE patients with active disease revealed that the elevated level of sFas dramatically decreased with improvement in clinical and laboratory findings, following corticosteroid therapy. We propose that the serum level of sFas can serve as an appropriate marker for evaluating SLE disease activity. Serum sFas is heterogeneous with respect to molecular structure, thus several mechanisms are involved in the generation of sFas.
T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4+ or CD8+ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8+ and CD4+ T cells from HIV-infected and HIV- subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8+ T cells from patients proliferated consistently less than controls, while responses from CD4+ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4+ and CD8+ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-gamma) in CD4+ T cells. Compared with controls, CD4+ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-gamma, IL-4 and IL-5, while CD8+ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4+ T cells from HIV+ subjects to various costimulatory pathways are relatively intact, whereas CD8+ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8+ T cell failure might contribute to viral and neoplastic complications of HIV infection.
Cytolytic induction of T cells requires both the T-cell receptor (TCR)-mediated antigenic stimulation and the CD28-mediated co-stimulatory signal. Blockade of the interactions between CD28 and its ligands, CD80 and CD86, prolongs the survival of allografts in some transplantation models. However, we found that allogeneic hepatocytes were completely rejected within 7 days after intrasplenic transplantation, even when treated with monoclonal antibodies (mAbs) against CD80 and CD86 (anti-CD80/86). Recent studies have shown that there are two main mechanisms of T-cell-mediated cytotoxicity, perforin-based and Fas-based ones. It has been shown that the liver is highly sensitive to induction of apoptosis by an agonistic anti-Fas mAb. We then investigated the role of the Fas/Fas ligand (FasL) system in the CD28-independent allogeneic hepatocyte rejection. With the anti-CD80/86 mAb treatment, hepatocytes from C57BL/6 lpr/lpr (B6 lpr) mice, which express little Fas antigen, could survive for 7 days after intrasplenic transplantation, and hepatocytes from C57BL/6 (B6) mice could also survive for 7 days in the spleen of C3H/ He gld/gld (C3H gld) mice, which express no functional FasL. CD28-independent induction of cytotoxicity against allogeneic hepatocytes was not observed when the effector cells were derived from C3H gld mice. These results indicated that the Fas/FasL system plays a critical role in the CD28-independent pathway of allogeneic hepatocyte rejection.
The interaction between CD28 and its ligands, CD80 and CD86, is crucial for an optimal activation of antigen-specific T cells. However, the requirement of CD80 or CD86 co-stimulation in Th2 cell differentiation and activation is controversial. Freshly isolated murine CD4+ and CD8+ T cells were incubated with P815 transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb. Both CD80 and CD86 co-stimulated the proliferation of CD4+ and CD8+ T cells at comparable time-kinetics and magnitude, but CD86 alone was able to co-stimulate IL-4 and especially IL-10 production in CD4+ T cells. In typical Th2-dependent immune responses elicited by Nippostrongylus brasillensis infection, the anti-CD86 mAb treatment but not the anti-CD80 mAb treatment efficiently inhibited antigen-specific IgE and IgG1 production, which was accompanied with the reduced IL-4 production. Our results suggest that CD86 co-stimulation plays a dominant role not only in the primary activation of Th2 cells but also in the secondary interaction between antigen-primed Th2 cells and B cells.
The CD28/B7 pathway provides a critical co-stimulatory signal for T cell activation. In the present study, we cloned rat CD80 and CD86 cDNA from a HTLV-1-transformed rat T cell line, Lewis-S1, expressing a high level of CTLA-4-Ig binding proteins. The predicted CD80 and CD86 polypeptides were composed of 321 and 313 amino acids respectively, and exhibited features common to human and mouse B7 family proteins. Both CD80 and CD86 mRNAs were abundantly detected in HTLV-1-transformed rat T cell lines but not in a thymic lymphoma cell line. To further explore the function of rat CD80 and CD86, we generated cDNA transfectants and anti-rat CD80 (3H5) and anti-rat CD86 (24F) mAb. Rat CD80 or CD86 transfectants exhibited a potent co-stimulatory activity for rat T cell proliferation, which was blocked by 3H5 and 24F mAb respectively. 3H5 or 24F immunoprecipitated a 80-90 or 90-100 kDa surface protein from Lewis-S1 cells. HTLV-1-transformed rat T cell lines expressed high levels of both CD80 and CD86 as estimated by staining with 3H5 and 24F, which acted co-stimulatory for allogeneic T cell activation as estimated by blocking with 3H5 and 24F. These mAb will be useful for investigating the pathophysiological functions of CD80 and CD86 in transplantation, autoimmune diseases and HTLV-1-associated pathologies in the rat system.