Modulation of the chaperone activities of Hsc70/Hsp40 by Hsp105alpha and Hsp105beta.

Hsp105alpha and Hsp105beta are stress proteins found in various mammals including human, mouse, and rat, which belong to the Hsp105/Hsp110 protein family. To elucidate their physiological functions, we examined here the chaperone activity of these stress proteins. Hsp105alpha and Hsp105beta prevented the aggregation of firefly luciferase during thermal denaturation, whereas the thermally denatured luciferase was not reactivated by itself or by rabbit reticulocyte lysate (RRL). On the other hand, Hsp105alpha and Hsp105beta suppressed the reactivation of thermally denatured luciferase by RRL and of chemically denatured luciferase by Hsc70/Hsp40 or RRL. Furthermore, although Hsp105alpha and Hsp105beta did not show ATPase activity, the addition of Hsp105alpha or Hsp105beta to Hsc70/Hsp40 enhanced the amount of hydrolysis of ATP greater than that of the Hsp40-stimulated Hsc70 ATPase activity. These findings suggest that Hsp105alpha and Hsp105beta are not only chaperones that prevent thermal aggregation of proteins, but also regulators of the Hsc70 chaperone system in mammalian cells.

Delayed clearance of zymosan-induced granuloma and depressed phagocytosis of macrophages with concomitant up-regulated kinase activities of Src-family in a human monocyte chemoattractant protein-1 transgenic mouse.

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

Participation of endogenously produced interferon gamma in interleukin 4-mediated tumor rejection.

To elucidate the molecular mechanism underlying IL-4-induced tumor rejection, we challenged mice with a mouse adenocarcinoma cell line, colon 26, genetically engineered to express constitutively IL-4 gene (colon 26/IL-4). Immunocompetent BALB/c mice rejected colon 26/IL-4 cells but not parental cells or cells transduced with a control gene (colon 26/control). Moreover, on rechallenge, parental cells and colon 26/control cells were rejected by normal BALB/c mice that had previously rejected colon 26/IL-4. However, both nude and severe combined immunodeficiency (SCID) mice failed to reject colon 26/IL-4 as well as parental or colon 26/control cells. In contrast, nude mice did reject colon 26/IL-4 after transfer of lymphocytes obtained from the draining lymph nodes of BALB/c mice injected with colon 26/IL-4. These results indicate that challenging mice with colon 26/IL-4 tumor cells resulted in the generation of memory cytotoxic T lymphocytes in the draining lymph nodes. At 3 days after the challenge, IFN-gamma, IL-12 p35, and p40 mRNA expression was selectively enhanced in the draining lymph nodes of mice bearing colon 26/IL-4 cells. Finally, mice deficient in the IFN-gamma gene did not reject colon 26/IL-4 cells. These results suggest that IL-4-induced memory cytotoxic T lymphocyte generation requires IFN-gamma production in the draining lymph nodes, in order to generate a protective immune response.

Intrathymic selection of NK1.1(+)alpha/beta T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad.

Generation and negative selection of NK1.1(+)alpha/beta T cell receptor (TCR)+ thymocytes were analyzed using TCR-transgenic (B10. D2 x DO10)F1 and (C57BL/6 x DO10)F1 mice and Rag-1(-/-)/DO10 mice, which had been established by breeding and backcrossing between Rag-1(-/-) and DO10 mice. Almost all T cells from these mice were shown to bear Valpha13/Vbeta8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-Ad. A normal proportion of the NK1.1(+) Valpha13/Vbeta8.2(+) thymocytes was generated in these mice. However, the actual cell number of both NK1.1(+) and NK1.1(-) thymocytes in I-Ad/d mice (positive selecting background) was larger than that in I-Ab/d mice (negative selecting background). Markedly low but significant proportions of NK1.1(+) Valpha13/Vbeta8.2(+) cells were detected in the spleens from I-Ad/d and I-Ab/d mice. It was shown that the splenic NK1.1(+) T cells of the I-Ab/d mice were anergized against stimulation through TCR. When (B10.D2 x DO10)F1 and (C57BL/6 x DO10)F1 mice were given cOVA, extensive or intermediate elimination of NK1.1(+)alpha/betaTCR+ thymocytes was induced in I-Ad/d or I-Ab/d mice, respectively. However, the clonal elimination was not as complete as that seen in the major NK1.1(-) thymocyte population. The present findings indicate that normal generation of NK1.1(+)alpha/betaTCR+ thymocytes occurs in the absence of Valpha14-Jalpha281 and that substantial negative selection operates on the NK1.1(+)alpha/betaTCR+ cells.

[Efficacy of combination of ondansetron injection and tablet in CAF-induced emesis in breast cancer patients].

Efficacy of combination of ondansetron injection and tablet on CAF (cyclophosphamide, adriamycin, 5-fluorouracil) induced emesis were investigated in 10 breast cancer patients (33 courses). Complete suppression rate of nausea or vomiting were approximately 75%, approximately 90% respectively for every treatment day. According to judgement criteria, antiemetic rate of approximately 100% was achieved during the study period. As to food intake of each treatment day, in approximately 70% of treatment courses was assessed as '(patient was) able to eat most of the meal'. Trend in emetic episodes and food intake in each patient receiving several courses of CAF therapy were evaluated. As a result, those patients experiencing nausea or vomiting or had effect on their food intake, were found to be in the similar condition in the following course (s) of CAF. No adverse drug reaction nor clinical laboratory test abnormalities due to ondansetron was observed. In this investigation, combination of ondansetron injection and tablet was shown to sufficiently suppress CAF-induced nausea and vomiting, and their efficacy was confirmed. Still, the study suggested that number of emetic episodes or degree of anorexia differs according to each individual. Therefore we regard additional administration of ondansetron or concomitant use of steroids should be considered when necessary.

[A study on mechanisms of thymic selection by intrathymic administration of antigenic peptides].

In the process of T cell differentiation thymocytes undergo positive and negative selections. The positive and negative selections result from interactions between the T cell antigen receptor (TCR) and self peptides presented by the major histocompatibility complex (MHC) molecules. Positive selection generates functional T cells restricted to the self-MHC. Negative selection eliminates or anergies T cells bearing self reactive TCR with high affinity to self peptides plus MHC. In this study, differentiation and selection of thymocytes were analyzed using bone marrow chimeras. It was demonstrated that differentiation of CD4+CD8+ thymocytes to CD4+ or CD8+ mature thymocytes occurred between 12 and 16 days post bone marrow transplantation. When pigeon cytochrome c 43-58 related peptide antigens were injected intrathymically 13 days after bone marrow transplantation, various changes were observed in resultant T cell repertoire. Intrathymic injection of peptides with high affinity to the MHC class II resulted in specific inhibition of T cell responses to the peptides, whereas injection of peptides with low affinity to the MHC induced no alteration. Furthermore, no modification of T cell responses was observed, when these peptides were administered after 14 days post BMT. The present findings demonstrate that when peptides with high affinity to the MHC class II molecules are administered intrathymically at a critical stage of thymocyte differentiation, negative selection is induced in a manner of one peptide vs one T cell clone.

Effects of non-major histocompatibility antigens on acute graft-versus-host reaction after allogeneic bone marrow transplantation.

In the present study using an experimental BMT system we analyzed the effects of disparity at non-MHC Ag including minor lymphocyte stimulatory-1a (Mls-1a) Ag on the acute GVH reaction (GVHR) induced by MHC class I Ag. Mismatch at MHC (class I) Ag alone did not induce clinically detectable acute GVHR in this model. However, BMT mice prepared with a combination of both class I and non-MHC Ag mismatches showed signs of clinical GVHR and various cytokines were produced by the spleen cells at an early stage (4 days) after BMT. Although no clinical GVHR was detected in BMT chimeras prepared with a non-MHC mismatched but MHC matched combination, large amounts of various cytokines were secreted by spleen cells. Cytokine production in the latter two kinds of chimeras paralleled the increase of Mls-1a reactive Vbeta6+ T cells in the host spleen. Marked cytokine production induced by Mls-1a Ag was confirmed by MLR. Thus, these cytokines appeared to be produced by T cells responding to Mls-1a (ie Vbeta6+ T cells) and to augment the T cell responses to MHC class I which resulted in clinically detectable GVHR in chimeras prepared with the combination mismatched at both MHC class I and non-MHC loci.

Influence of graft versus host reaction on the T cell repertoire differentiating from bone marrow precursors following allogeneic bone marrow transplantation.

When lethally irradiated AKR (Mls-1a) mice were reconstituted with bone marrow (BM) cells plus a small number (0.5%) of mature T cells from allogeneic B10.AQR or B10 (Mls-1b) mice and minor GVHR was induced in the recipients, almost complete donor chimerism was accomplished in the early stages after reconstitution. By contrast, in irradiated AKR mice reconstituted with T cell-depleted BM cells alone from B10 or B10.AQR mice, radio-resistant T cells of recipient origin persisted for a relatively long period in peripheral lymphoid tissues. In this paper the influence of residual T cells in the chimeric mice on generation of the T cell repertoire derived from donor BM is discussed. It will be demonstrated that the recipient (AKR) T cells are capable of producing Mls-1a antigens (Ag) after lethal irradiation in vivo. These recipient T cells eventually induce clonal elimination of Mls-1a reactive V beta 6+, V beta 8.1+ and V beta 9+ T cells derived from developing thymocytes of donor BM origin. The Mls-1a reactive T cells are not eliminated in GVHR chimeras in which recipient T cells are absent. However, V beta 5+ T cells reactive to I-E plus Etc-1 Ag are deleted in the chimeras undergoing GVHR. These results indicate that recipient cells which produce tissue-specific antigens (tolerogens) should be taken into consideration when generation of the T cell repertoire of donor origin following allogeneic BM transplantation is investigated.

Generation of NK1.1+ T cell antigen receptor alpha/beta+ thymocytes associated with intact thymic structure.

The development of T cells within the thymus is largely dependent on intact cortical and medullary epithelial cells. However, it has been reported that positive selection of natural killer antigen 1.1+ (NK1.1+) T cell antigen receptor (TCR)-alpha/beta+ thymocytes recently identified among CD4+8- and CD4-8- subpopulations is attributable to major histocompatibility complex class Ib ligands expressed on bone marrow (BM)-derived components in the thymus. In the present study, we investigated generation of NK1.1+ TCR-alpha/beta+ cells in the thymus of the aly/aly mouse which lacks lymph nodes and Peyer's patches and shows abnormalities of thymic and splenic structure. We found that the proportion of the NK1.1+ TCR-alpha/beta+ thymocytes was extremely low in these mice as compared with aly/+ and normal C57BL/6 mice. Thymic reconstitution by BM cells from aly/+ mice that possess a normal population of NK1.1+ TCR-alpha/beta+ cell population did not restore the NK1.1+ TCR-alpha/beta+ cell population in the thymus of lethally irradiated aly/aly mouse. When deoxyguanosine-treated fetal thymi from (B6 x B10.G)F1 mice were transplanted to aly/aly mice that had been thymectomized and reconstituted with BM cells of aly/aly mice, normal proportions of the NK1.1+ TCR-alpha/beta+ thymocytes were present in the thymus grafts. These findings demonstrate that the development of NK1.1+ TCR-alpha/beta+ thymocytes is accomplished under the influence not only of BM-derived components, but also of irradiation-resistant or deoxyguanosine-resistant components and an intact microenvironment of the thymus.

A promiscuous T cell hybridoma restricted to various I-A molecules.

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-Ag7) mice, we established a T cell hybridoma specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-Ag7, but also I-Ad, s, u and v. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic alpha and polymorphic beta chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.