Human CD4 helper T cell activation: functional involvement of two distinct collagen receptors, 1F7 and VLA integrin family.

In the present study, we showed that activation of human CD4 T cells can be induced by anti-CD3 and collagen in a serum-free system. This activation was inhibited by the addition of peptides containing the RGD or Gly-Pro-X sequences. Significantly, we demonstrated that both the 1F7 (CD26) structure and the VLA integrin family, particularly the VLA-3 complex, contribute to the functional interaction between collagen and CD4 cells since anti-1F7 and anti-VLA-3 specifically inhibited this collagen-induced CD4 cell activation. Biochemical studies showed that the 1F7 structure is not a member of the VLA integrin family. These results thus indicated that two different families of antigens serve as functional collagen receptors for CD4 T cell activation.

A monoclonal antibody (8H3) that binds to rat T lineage cells and augments in vitro proliferative responses.

A murine monoclonal antibody, designated 8H3, recognizes a cell surface antigen expressed exclusively on rat T lineage cells. 8H3 antibody immunoprecipitated 180-, 120-, and 90-kD components from rat thymocytes as well as splenic T cells under nonreducing conditions. 8H3 antibody specifically inhibited the binding of thymocytes to fibronectin. Furthermore, binding of rat thymocytes to immobilized synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys-BSA was inhibited by 8H3 antibody as was Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by Gly-Arg-Ala-Asp-Ser-Pro-Lys or Gly-Arg-Gly-Glu-Ser-Pro. Crosslinking of 8H3 antigen on double-negative thymocytes and adult thymocytes, as well as splenic T lymphocytes by 8H3 antibody and F(ab')2 fragments of goat antibodies to mouse immunoglobulin, led to an increase in the concentration of cytoplasmic free Ca2+ due to the release of Ca2+ from intracellular stores as well as the influx of Ca2+ from extracellular sources. Expression of interleukin 2 receptor and subsequently cell proliferation was observed upon incubation of thymocytes and splenic T cells with 8H3 antibody. Furthermore, 8H3 antibody induced the proliferation of double-negative thymocytes. These data collectively indicated that a cell surface antigen, 8H3, is involved in not only cell adhesion but also involved in the expression of immature as well as mature thymocytes.

Biochemical characterization of CD26 (dipeptidyl peptidase IV): functional comparison of distinct epitopes recognized by various anti-CD26 monoclonal antibodies.

In this paper, we performed further biochemical characterization of the CD26 antigen, as defined by the mAbs in anti-1F7 and anti-Ta1, in order to clarify the observed functional differences among these mAbs. For this purpose, we developed a mAb, anti-5F8, which recognizes yet another epitope on the CD26 antigen different from that recognized by anti-1F7 and anti-Ta1 and compared their respective effect on T cell activation as well as the structures recognized by these mAbs. Functionally, anti-5F8 did not exhibit a comitogenic effect on T cell activation via the CD3 and CD2 pathways. Peptide mapping studies suggested that the 110 kDa molecules precipitated by these mAbs are identical. We showed that the 110 kDa CD26 structure on human T cells is composed of a family of heterogeneous molecules, as determined by isoelectric focusing studies. In addition, we demonstrated that the CD26 antigen has a DPPIV enzyme activity and this enzyme activity is found only on the principal basic structure of CD26 but not on the additional acidic structures. Biochemical studies also revealed that these mAbs recognized distinct epitopes on the CD26 antigen. Pulse-chase studies showed the the 1F7 epitope was found on both the immature (100 kDa) and mature (110 kDa) forms of the CD26 antigen. On the other hand, the Ta1 and 5F8 epitopes were expressed mainly on the mature form of the CD26 antigen. Moreover, anti-IF7 consistently precipitated an additional 43 kDa molecule in association with the principal 110 kDa molecule. Taken together, these data suggested that the additional 43 kDa structure or the distinct epitope recognized by anti-IF7 may play a role in human T cell activation via the CD3 and CD2 pathways.

Discordant expression of myeloid antigens and myeloperoxidase in a case of t(8;21) positive AML expressing CD7.

We describe a case of acute myeloid leukemia (AML) showing myeloperoxidase (MPO)-positive and myeloid antigens negative. Although the leukemic cells showed few granules in May-Grünwald Giemsa staining, cytochemical MPO staining revealed that most of the blast cells strongly reacted with MPO. The leukemic cells did not express myeloid antigens (CD13, CD33), nor B-lymphoid or T-lymphoid antigens on the cell surface using flow cytometry, however. The cells did express CD34 and CD7. Discordant expression of MPO and myeloid antigens was also confirmed by electron microscopic MPO staining and by immunocytochemistry using a streptoavidin-biotin alkaline phosphatase labeling technique. Cytogenetic studies showed 46, XX, t(8;21) (q22;q22), del (9) (q22) in the bone marrow cells. In addition, AML1/ETO chimeric mRNA was detected from these cells. We summarize eight reported cases of MPO positive and myeloid antigens negative AML. Five of nine cases including our case had the same chromosomal abnormality of t(8;21) (q22;q22) and showed better prognosis than the other cases.

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma.

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensitized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Immunochemical characterization of monoclonal protein in the serum of a patient with Waldenstr]om's macroglobulinemia showing both pyroglobulin and cryoglobulin properties.

A patient with Waldenstr]om's macroglobulinemia whose serum demonstrated properties of both pyroglobulin and cryoglobulin was studied. A monoclonal (M) protein in the serum of the patient was identified by immunoelectrophoresis (IEP) and immunofixation electrophoresis (IFE) as IgM-lambda. The M-protein was separated by gel permeation high-performance liquid chromatography (HPLC). Cryoglobulin in the serum was isolated by the method of cold precipitation. The cryoglobulin was identified as IgM-lambda type by IEP and was the same M-protein as that which occurred in this patient's serum. The purified cryoglobulin also had the properties of a pyroglobulin. Neither property disappeared following pretreatment with 2-mercaptoethanol (2-ME), urea, and Triton X-100 detergent, and deglycosylation with N-glycanase (E.C. 3.5.1.52). We suggest that these abnormal properties were caused by the molecular abnormality of the IgM-lambda M-protein.

[Multiple myeloma with a solitary osteosclerotic legion on seventh cervical vertebra].

The osseous manifestation of multiple myeloma is well known as the osteolytic or osteoporotic feature. On the other hand, there are rare cases of general osteosclerotic manifestation as myeloma variants. We report a case of multiple myeloma with solitary osteosclerotic legion in the cervical vertebra. A 60-year-old-man was admitted with paralysis of both arms. The cervical roentogenogram showed the osteosclerotic change of the seventh cervical vertebra. The pathological study of surgical bone biopsy from the vertebra revealed osseous and severe fibrotic change and accumulation of plasma cells in the residual bone marrow. Clusters of plasma cells were also observed in the bone marrow of ileac bone. In addition, IgG-lambda type M protein was seen in the serum. Therefore we diagnosed this case as the osteosclerotic multiple myeloma. We then analyzed the cytokines known to influence bone formation, and found that the bone marrow serum TGF-beta and PDGF levels were increased compared with normal control. These results may suggest that the preferential increase of osteosclerotic cytokines caused the osteosclerotic changes of bone marrow.

[Reevaluation of the surgical treatment of thyroid nodules diagnosed as cancer postoperatively].

Reevaluation was carried out on the surgical treatment for clinically benign thyroid nodules. One hundred and thirty-seven patients underwent conservative resections of the thyroid because of preoperative impression of benign nodules at the First Department of Surgery, Nagoya University Hospital from 1970 to 1984. Permanent paraffin sections of the resected specimen revealed that the nodules in 4 patients were intrathyroidal cancer (3 papillary and 1 follicular) and the nodules in the other 8 patients were associated with an unsuspected small (3 approximately 15 mm) thyroid cancer. All those patients underwent either lobectomy or subtotal thyroidectomy at the initial surgery except for one patient in whom enucleation was performed. No further intervention was carried out when the definitive diagnosis was made, except for one patient in whom the enucleation was followed by lobectomy. No neck dissection was attempted in all of them. Patients have been living and well without evidence of recurrence for 3 to 11 years and one died of unrelated disease. From these results, it is recommended that lobectomy is the least requirement in treating clinically benign nodule and that, when paraffin section reveals the tumor is intrathyroidal cancer, no further surgery is justified unless there are other foci of cancer in the resected lobe.

Etoposide-containing conditioning regimen reduces the occurrence of hemophagocytic lymphohistiocytosis after SCT.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages that secrete high amounts of inflammatory cytokines. HLH occurring after SCT is difficult to diagnose. It is characterized by severe clinical manifestations and high mortality. Despite current therapeutic approaches, outcomes remain poor. We analyzed the incidence and risk factors of HLH after SCT and the response to treatment and prognosis of 554 patients with HLH after SCT. The cumulative incidence of HLH after SCT was 4.3% (24/554). Use of etoposide in the conditioning regimen was only factor that reduced HLH after SCT (P=0.027). All patients who received autologous transplantation were successfully treated. Patients with liver dysfunction (for example, high total bilirubin level, prolonged prothrombin time and high level of fibrinogen degradation products) had a poor response to treatment for HLH. Physicians should be cautious of HLH, while not using etoposide for conditioning regimen.

Inactivation of Escherichia coli by O- water.

AIMS: To clarify the effects of O(-) (atomic oxygen radical anion) water on the viability and morphological alteration of Escherichia coli. METHODS AND RESULTS: O(-) water (OW) was prepared by bubbling of O(-)/argon (Ar) flux into deionized water. O(-) and hydrogen peroxide (H(2)O(2)) in the resultant OW were analysed by electron paramagnetic resonance and ultraviolet (UV) absorption spectroscopy. The population of E. coli treated by a typical OW of pH 4.30 +/- 0.20 [(2.5 +/- 0.8) x 10(-3) mmol l(-1) O(-); 0.5 +/- 0.2 mmol l(-1) H(2)O(2)) was reduced by more than 3 log CFU ml(-1) within 60 min at 30 degrees C. Through scanning electron microscopy observation, the OW-treated cells appeared dramatically collapsed. The release of nucleic acid induced by OW was identified by UV absorption spectroscopy. CONCLUSIONS: O(-) water can result in inactivation of E. coli, nucleic acid release and cellular damage under the controlled laboratory conditions in excess of 15-30 min. Reactive oxygen species may play an important role in the inactivation process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study first revealed that OW could inactivate E. coli, which may be potentially useful in developing a novel approach for the microbial decontamination of food, water or heat-sensitive material.

[Immunohistochemical studies on the thyroid C-cells in primary hyperparathyroidism].

This study was conducted in order to establish whether C cells, which are responsible for secretion of calcitonin within the thyroid gland, change either in volume or morphology under conditions of chronic hypercalcemia in primary hyperparathyroidism. Out of 106 primary hyperparathyroid patients undergoing surgery, in 11 cases the thyroids were excised and examined for changes in the C cell. As a control group we used thyroids removed in another 14 cases undergoing thyroidectomy or laryngectomy. Calcitonin in the C cell was observed by optical microscope after immuno staining using the indirect peroxidase-labeled antibody technique. C cells are not evenly distributed within the thyroid. However, there is excellent positive correlation (p less than 0.001) between the C-cell index, which is the average of two tissue samples excised from the area at the border between the upper 1/3 and middle 1/3 of the thyroid lobe (the area where most C cells are found), and the total number of C cells. The C-cell index can thus be used as an indicator of the total number of C cells in the thyroid. The number of C cells decreased (p less than 0.01) as the level of calcium in serum increased. In patients with primary hyperparathyroidism, this decrease in C cells was significantly greater (p less than 0.025) than in the controls. Focal C cell hyperplasia and diffuse C cell hyperplasia were present in both the control group and primary hyperparathyroid group, but there was no significant difference between the two groups as to the frequency of occurrence. For both these conditions the rate of occurrence was considered within normal ranges for C cell morphology. We concluded that the decrease in C-cell count in primary hyperparathyroidism patients with chronic hypercalcemia is due to consumption of calcitonin in the C cell.

A signal transduction through a unique rat T cell specific antigen, 8H3.

A signal transduction was detected in various rat T cells by cross-linking of 8H3 antigen by 8H3 antibody. A rat T cell proliferative response induced by 8H3 antibody is dependent on the presence of adherent cells. When spleen cells were cultured in the presence of 8H3 antibody, only CD4-positive T cell proliferation was induced. These proliferative CD4-positive T cells express rat interleukin 2 receptor, alpha chain. Cross-linking of 8H3 antigen resulted in an increase in cytoplasmic free Ca2+ in T cells and phosphorylation of a 120 kDa component of the 8H3 antigen. It should be noted that cross-linking of 8H3 antigen together with CD4 antigen but not with CD8 antigen by suboptimal doses of 8H3 and corresponding antibodies initiated the mobilization of [Ca2+]i. Furthermore, physical association of 8H3 antigen with TCR was demonstrated by comodulation of these two molecular complexes in some T cells. Thus, 8H3 antigen is involved in rat T cell transmembrane signal transduction.

The 1A4 molecule (CD27) is involved in T cell activation.

We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation.