Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.

Ligation of a B lymphocyte surface immunoglobulin (sIg) antigen receptor (AgR) by its specific Ag ligand initiates a signaling pathway that culminates in B cell activation. However, many events of this pathway have not been elucidated. Here we present three novel findings that demonstrate directly that AgR-mediated signaling in B cells functions by the p21ras/ras.GAP-dependent pathway. First, stimulation of TA3 7.9 Ag-specific murine B lymphoma cells for 2 min with either Ag or F(ab')2 anti-IgM induces p21ras activation as measured by an increase in the GTP/GDP ratio of its bound nucleotides. This activation of p21ras does not occur via a change in its guanine nucleotide exchange rate. Second, Ag stimulation results in the inhibition of activity of p120 ras.GAP, a protein that regulates p21ras activation. Tyrosine phosphorylation of ras.GAP occurs within 1 min after Ag stimulation but is no longer detectable at 20 min after stimulation, at which time ras.GAP activity remains inhibited. Thus, tyrosine phosphorylation of ras.GAP is not required for the inhibition of its activity. Third, despite the role proposed for a ras.GAP-associated p190 protein in the control of ras.GAP activity in B cells, p190 was not detectable either in anti-ras.GAP immunoprecipitates of [35S]methionine labeled lysates of Ag-stimulated or -unstimulated 7.9 cells or as a tyrosine phosphoprotein in Western blots of anti-ras.GAP immunoprecipitates of Ag-stimulated 7.9 cell lysates. Inasmuch as the TA3 7.9 B lymphoma is representative of a mature, sIgM-bearing B cell, our observations raise the intriguing possibility that the capacity of p190 to associate with ras.GAP and regulate the activities of ras.GAP and p21ras in a B cell is dependent on the stage of differentiation of the B cell.

Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

Human beta-defensin-3 induction in H. pylori-infected gastric mucosal tissues.

AIM: To examine human beta-defensin-3 (hBD-3) expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori infection for better understanding the innate immune response to H. pylori. METHODS: We used reverse transcription-polymerase chain reactions and immunohistochemistry to examine hBD-3 expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori. Effects of hBD-3 against H. pylori were also evaluated. RESULTS: The mean mRNA expression of hBD-3 in H. pylori-positive specimens was significantly higher than that in H pylori-negative specimens (P = 0.0002, Mann-Whitney). In addition, unlike uninfected samples, 8 of 15 (53.33%) infected mucosal samples expressed hBD-3 protein. H. pylori dose-dependently induced mRNA expression of hBD-3 in MKN45 cells, an effect inhibited by adding anti-toll-like receptor (TLR)-4 antibody. HBD-3 protein completely inhibited H. pylori growth. CONCLUSION: Our results suggest that like hBD-2, hBD-3 may be involved in the pathophysiology of H. pylori-induced gastritis.

Regulation of BCR- and PKC/Ca(2+)-mediated activation of the Raf1/MEK/MAPK pathway by protein-tyrosine kinase and -tyrosine phosphatase activities.

Ligation of the B cell Ag receptor (BCR) activates a protein-tyrosine kinase (PTK) and CD45 protein-tyrosine phosphatase (PTPase)-dependent signaling cascade that results in the activation of Ras. This pathway of Ras activation can operate independently of protein kinase C (PKC) activity. Activation of Ras may lead to two distinct Ras-dependent pathways involving either a Raf1/MEK/MAPK module or a MEKK/SEK/SAPK module; however, it is unclear as to how Ras controls the independent activation of either of these pathways. We have used genistein and phenylarsine oxide (PAO) as inhibitors of PTK and PTPase, respectively, to investigate whether they regulate the BCR- and Ca2+/PKC-dependent activation of the Ras/Raf1/MEK/MAPK module. Assays of phosphotransferase activities conducted with Ag (TNP6-OVA)-specific 7.9 murine B lymphoma cells demonstrated that BCR-mediated stimulation of the Raf1/MEK/MAPK module is controlled by PTK and PTPase activities. An elevation in [Ca2+]i was required to optimally activate Raf1 and MEK through the BCR. However, when signaling through the BCR was bypassed by direct stimulation of the Raf1/MEK/MAPK module via a rise in [Ca2+]i and phorbol ester-induced PKC activation, the phosphotransferase activities of Raf1, MEK and MAPK were still regulated in a PTK-dependent manner that was also partially sensitive to the PTPase inhibitor PAO. Thus, at least two alternate routes, i.e. a BCR/PTK/Ras-dependent route and another PKC/Ca(2+)-dependent route, may converge at the level of Raf1 for activation of the Raf1/MEK/MAPK module in B cells.

A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.

Near-infrared FT-Raman spectra of the rat brain tissues.

Near-infrared Fourier transform (FT) Raman spectroscopy was applied to brain tissues in situ. The spectra were obtained from the cerebral cortex, white matter of the cerebrum, caudate-putamen, thalamus, synaptosomal fraction, and myelin fraction. High-quality Raman spectra in the 400 to 2940 cm-1 range were measured without interference of autofluorescence. Common spectral bands were assigned. The ratios of the intensity at 1664 (amide I), 1442 (CH2 deformation), 2885 (CH2 asymmetric stretching), 2938 cm-1 (CH3 symmetric stretching) could be used for differentiation between the gray and white matters.

Near-infrared Fourier transform Raman spectroscopic study of cornea and sclera.

Near-infrared excited Fourier transform Raman spectroscopy was applied in situ on intact rabbit cornea and sclera. High quality Raman spectra were obtained noninvasively from these tissues. The characteristic bands assigned to proline and hydroxyproline appeared in the region of 800-1000 cm-1. Raman spectra of purified type I collagen and other components of the cornea and sclera were also determined. The main elements of the spectra of the cornea and sclera originated from the collagen proteins, and other components such as proteoglycan contributed little. Near-infrared excited Fourier transform Raman spectroscopy can be used for biophysical study of the cornea and sclera in situ or in vivo.

[Clinical evaluation of meropenem in the pediatric field].

A clinical study on a new carbapenem antibiotic, meropenem (MEPM), was carried out in acute pediatric infections. MEPM was administered to 8 patients including 3 patients with acute pneumonia, 2 with cervical lymphadenitis, 1 with acute tonsillitis, and 1 with cellulitis and 1 with sepsis. The overall efficacy rate was 100%. As an adverse reaction, diarrhea was observed in 1 patient. In clinical laboratory tests 1 patient was found to have S-GPT elevation which normalized after discontinuation of MEPM. MEPM appears to be effective and safe drug for pediatric acute infections.

[Clinical studies on cefdinir in pediatric infections].

Pharmacokinetic, bacteriological and clinical studies on cefdinir (CFDN), a newly developed oral cephalosporin, were performed on children with infections. The pharmacokinetics was examined in 3 patients. The peak plasma concentrations were 1.97 micrograms/ml, 0.84 microgram/ml and 1.67 micrograms/ml in the 3 patients. The 0 to 6 or 8-hour urinary excretion rates were 22.2%, 18.1%, and 32.7%, respectively. These results were similar to those in adult patients. Clinical response to CFDN was evaluated in 21 patients, 4 patients with pharyngitis (an efficacy rate of 100%), 7 with tonsillitis (85.7%), 1 with bronchitis (excellent), 1 with pneumonia (fair), 6 with scarlet fever (100%), 1 with staphylococcal scaled skin syndrome (good) and 1 with urinary tract infection (good). Thus, an overall efficacy rate of 90.5% was achieved. With regard to microbiological effect on pathogens, 14 of the 15 strains identified as pathogens were eradicated, with an eradication rate of 93.3%. The safety was evaluated in a total of 23 cases. Diarrhea, elevated eosinophil count and elevated S-GPT were observed in one patient each. The side effect and abnormalities in laboratory tests were not serious, however. It was concluded that CFDN, with its excellent antibacterial effect, was an efficacious and safe drug for the treatment of pediatric infections.

[Clinical results of a rokitamycin dry syrup in pediatric infections].

Clinical efficacies of a new macrolide antibiotic, rokitamycin (RKM, TMS-19-Q), were studied in acute pediatric infections. Responses to the RKM administration were evaluable in 62 out of 68 patients consisted of 7 patients with pharyngitis (efficacy rate of 85.7%, 6/7 patients), 4 with bronchitis (25.0%, 1/4), 9 with tonsillitis (100%, 9/9), 13 with mycoplasmal pneumonia (100%, 13/13), 13 with hemolytic streptococcal infections (92.3%, 12/13), 14 with pneumonia (57.1%, 8/14), one with pertussis (100%, 1/1) and another with Chlamydia pneumonia (100%, 1/1) thus an overall efficacy rate of 82.3% was achieved. Urticaria was observed in one of the patients as an adverse reaction to the drug, while abnormal laboratory test results were noted in 3 patients, but none of such changes were severe. The drug, even when administered in combination with a theophylline preparation, exerted no effects on the serum concentration of the latter.

[Complex karyotypic abnormality in an aged patient with acute myeloid leukemia (M 2)].

A 74-year-old man was admitted on November 1986 because of general fatigue. His peripheral blood showed pancytopenia without immature cells since December 1985. Hematological data showed RBC 150 X 10(4)/microliter, PLT 7,000/microliter, WBC 12,000/microliter with 93.6% leukemic cells. The bone marrow smear revealed NCC 14.5 x 10(4)/microliter with 76% leukemic cells. The leukemic cells were characterized by faint staining with peroxidase stain and strong positivity for CD 13 antigen determined with immunoperoxidase method and flow cytometric analysis. The chromosomal analysis of tumor cells represented as follows: 44, XY, -3, -4, -9, -20, 2q+, 6p-, 7q-, 12q+, +2 mar. Although remarkable reduction of leukemic cells in peripheral blood was obtained one month after initiation of 19-days intravenous continuous infusion of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), he suffered from severe systemic candida infection with severe leukopenia and died. Not only advanced age but also complex karyotypic abnormality would contribute to failure of treatment in this case. The significance of complex karyotypic abnormality in acute non-lymphocytic leukemia in discussed based on the current literature.

[Sustained long-term complete remission in an elderly aplastic anemia patient after cessation of combined therapy consisting of granulocyte-colony stimulating factor and erythropoietin].

An 83-year-old woman received a diagnosis of moderate aplastic anemia in November 1990. Immunosuppressive therapy consisting of anti-lymphocyte globulin combined with high-dose corticosteroids was effective until pancytopenia developed in August 1993. The patient was hospitalized again and recurrence of aplastic anemia was diagnosed on the basis of hematologic findings, including RBC 129 x 10(4)/microliter, Hb 5.5 g/dl, Ret 23,200/microliter, WBC 2,200/microliter with 27% neutrophils, platelets 2.2 x 10(4)/microliter, and hypoplastic bone marrow. Recombinant human granulocyte-colony stimulating factor (G-CSF) of 125 micrograms/day combined with recombinant human erythropoietin (EPO) of 6,000 U/day were started in November 1993. The doses of G-CSF and EPO were increased to 250 micrograms/day and 12,000 U/day, respectively. We stopped combination therapy in March 1995, after trilineage hematopoietic cell recovery had been achieved. Complete recovery in peripheral blood was sustained for more than 2 years despite the termination of G-CSF and EPO therapy. Combination therapy with G-CSF and EPO may be safe and effective for elderly patients with aplastic anemia when the choice of therapy is limited.

[Effectiveness of antilymphocyte and antithymocyte globulins for patients with severe aplastic anemia and pure red cell aplasia--analysis of immunologic parameters of peripheral lymphocytes concerning ALG and ATG therapy].

Four patients with severe aplastic anemia and one patient with pure red cell aplasia (PRCA) were treated with antilymphocyte and antithymocyte globulins. One patient in aplastic anemia who achieved good response by ALG administration had a possible diagnosis of myelodysplastic syndrome. ATG was administered to only one case of aplastic anemia and ALG was administered to the remainder. In the result, three out of 4 patients with aplastic anemia and one patient with PRCA achieved good response without severe side effects. Three patients with aplastic anemia had high CD4/CD8 ratio in their peripheral lymphocytes. This ratio normalized after ALG therapy in effective cases, but not in ineffective case. Natural killer activity elevated after ALG therapy in two effective cases of aplastic anemia and PRCA, but not in one ineffective case of aplastic anemia.

[Testicular involvement in IgD multiple myeloma].

A 65-year-old man was admitted to our hospital in September 1997 because of back pain and renal dysfunction. He was diagnosed as having multiple myeloma due to the presence of IgD lambda monoclonal gammopathy and diffuse infiltration of plasma cells in the bone marrow. The patient achieved a partial response to DMVM-IFN-alpha combination therapy. His condition worsened in December 1998, but was ameliorated by VAD therapy. The patient's left testis became enlarged in December 1999, and an orchiectomy was performed. The normal testicular cells had been entirely displaced by myeloma cells comprising typical plasma cells and large lymphoid cells. Pleural, mediastinal, spinal, and right testicular involvement with myeloma subsequently developed. Despite attempts to treat the patient with more than one type of combination therapy, his condition worsened progressively, and he died in June 2000. Reports of IgD myeloma with testicular involvement are rare. The histopathology of our patient's resected testis, i.e. the two myeloma cell-plasmacytoid and lymphoid cell components showing differential immunostaining, was unique.

[Improvement with interferon-alpha therapy and splenectomy in hairy cell leukemia of European-American type].

A 61-year-old man was admitted to our hospital in April 18, 1988, with dyspnea and gingival bleeding. Physical examination revealed marked splenomegaly, and peripheral blood showed severe pancytopenia with 38% abnormal mononuclear cells. The abnormal cells were characterized by a hairy appearance under a phase contrast microscopy, and strong tartrate-resistant acid phosphatase activity. These cells reacted with CD19, CD25 and CD11c monoclonal antibodies by the immunostaining method. Bone marrow aspiration failed and bone marrow biopsy revealed diffuse proliferation of hairy cells (HC) with moderate fibrosis. In addition, the staining pattern of HC peroxidase is similar to that found in megakaryocyte series. He was diagnosed as HCL of the European-American type based on these findings. Interferon (IFN)-alpha was administered at a daily dosage of 3 x 10(6) IU by intramuscular injection. Although splenomegaly and hematological conditions improved gradually, he received splenectomy because of his incomplete hematological improvement. Normalization of peripheral blood cell counts and a marked decrease of HC in bone marrow were obtained. Tubuloreticular structure and tubular confronting cisternae were seen in peripheral mononuclear cells during IFN therapy.

[Severe aplastic anemia successfully treated with cyclosporin A and prednisolone].

A 7-year-old boy was admitted to our department complaining pale face and subcutaneous bleeding in August, 1987. Peripheral blood analysis showed pancytopenia of WBC 2,600/microliter, RBC 148 x 10(4)/microliter and platelets 5,000/microliter. Bone marrow biopsy revealed hypocellularity. Granulocytes 104/microliter, reticulocytes 4,290/microliter and platelets 5,000/microliter were compatible with the diagnosis of severe aplastic anemia based on the criteria of the Ministry of Public Welfare in Japan. Prednisolone (PDN) was initially indicated and bolus methylprednisolone, metenolone and ALG therapy followed with no hematological improvement. Fifteen months after admission, in addition to 0.5-1 mg/kg/day of metenolone, Cyclosporin A (CyA) was started at a dose of 12 mg/kg/day for a week and 6 mg/kg/day thereafter. After a week from administration of CyA, 1 mg/kg/day of PDN was given because his bleeding tendency became worse. But this combination was complicated with liver damage and hyperglycemia to discontinue both drugs. These adverse effects were subsided within 7 days by cessation of the drugs. CyA was started again at a dose of 6 mg/kg/day without any response for 4 weeks. Then PDN was added together at a reduced dose of 0.5-1 mg/kg/day. Hematological response was obtained promptly. Granulocytes reached 1,500/microliter, hemoglobin 10.2 g/dl and platelets 26,000/microliter after 3 months of therapy. Afterward the patient became transfusion independent. The most effective method of CyA administration for aplastic anemia is still controversial. Alternative use of CyA, considering combination of steroids or anabolic steroids, in patients who failed to respond to conventional immunosuppressive treatments should be further investigated.

[Effect of blood conservation in open-heart surgery: a comparison of 3 different methods].

Conventional blood conservation techniques have been insufficient to decrease blood transfusion requirement in open-heart surgery. Blood conservation and erythropoietin administration were performed to avoid homologous blood transfusion. Intraoperative autotransfusion has been routinely used in cardiac operations with cardiopulmonary bypass in our hospital. To evaluate the effect of conservation techniques, 286 patients were divided into four groups. In group I (23 patients), autologous whole blood was drawn and saved one to two weeks before operation. In group II (50 patients), erythropoietin preparation was given subcutaneously once a week and autologous blood conservation was also performed in the same manner as group I. In group III (48 patients), intra-operative hemodilutional autologous blood transfusion was performed. In group IV, as a control group (165 patients), only intra-operative autotransfusion was used. Homologous blood transfusion was avoided in 83% of group I patients, in 90% of group II, in 82% of group III, and 29% of group IV. In addition, in group II the hemoglobin value at the time of discharge was significantly higher than those of other groups (p < 0.05-0.01). Thus, conventional blood conservation techniques plus subcutaneous administration of erythropoietin was very effective to increase the rate of "non-blood" open-heart surgery.

[Bladder irrigation with prostaglandin E2 in cyclophosphamide-induced hemorrhagic cystitis].

A four-year-old boy with stage IV neuroblastoma was treated using the group study protocol of the Tohoku area for advanced neuroblastoma, consisting of DTIC, CPA, VCR, CDDP and VM-26, as a result of which had obtained complete remission. However, he had severe hemorrhagic cystitis after administration of CPA. He was treated with the usual therapy, but symptoms such as hematuria, pollakiuria and miction pain were not improved. We then tried bladder irrigation with prostaglandin E2. Half a milligram of PGE2 in 100 ml of physiological saline solution was instilled into the bladder and left in situ for 3 hours. The patient was free of symptoms on the day following the therapy. Local therapy with PGE2 thus seems very useful for cyclophosphamide-induced cystitis.

[Characteristic of cell components in bronchoalveolar lavage fluid (BALF) in patients with bronchial asthma classified by clinical symptoms].

The characteristics of cell components in BALF were examined in three groups of 104 patients with bronchial asthma, classified by clinical symptoms. 1. The frequency of neutrophils in BALF was significantly higher in patients with hypersecretion type (Ib) and in cases with bronchiolar obstruction type (II) than in cases with simple bronchoconstriction type (Ia). 2. A significantly higher proportions of neutrophils in BALF was observed in atopic asthmatics with type Ib than in non-atopic asthmatics with type Ib. 3. More frequency with moderate or high grade of eosinophilia in BALF were observed in cases with type Ib, whether they were atopic or non-atopic type. 4. More frequency with moderate or high grade of neutrophilia in BALF were observed in atopic cases with Ib and in both atopic and non-atopic cases with II. 5. Cases with moderate and high grade of both eosinophilia and neutrophilia in BALF were more frequent in atopic cases with Ib. The results suggest followings--1) both eosinophils and neutrophils participate in hypersecretion of type Ib in atopic cases, and only eosinophils in non-atopic cases. 2) neutrophils participate in bronchiolar obstruction of type II, whether they are atopic or non-atopic cases.

[The role of p21ras oncoprotein in signal transduction mediated through B cell antigen receptor (BCR)].

Ligation of B cell antigen receptor (BCR) with antigen or anti IgM leads to enter cells into the proliferation and differentiation to produce specific Ig. Protein tyrosine kinase (PTK) and CD 45 protein tyrosine phosphatase (PTP) both are involved in the early phase of BCR-mediated B cell signaling. We have investigated the role of p 21ras(ras) in B cell signal transduction using TNP-specific TA3 7.9 murine B cells. B cell stimulation with either TNP6-OVA(Ag) or anti-IgM resulted in rapid accumulation of GTP-bound(active) ras as well as induction of a number of tyrosine phosphorylated substrates. The accumulation of GTP-bound ras was blocked by the treatment with either PTK inhibitors(genistein) or PTP inhibitors(PAO), suggesting that BCR-mediated ras activation is regulated by both PTK and PTP including CD 45. As phosphorylation on tyrosine residues of Lyn, Fyn, and Blk protein tyrosine kinases occurred upon BCR stimulation, these PTKs may be candidates being involved in an induction of not only tyrosine phosphorylation of substrates but also p 21ras activation. Furthermore we found that rasGAP activity is suppressed following Ag stimulation, accompanied by the phosphorylation on tyrosine of rasGAP and its associated protein p 62. These data indicate that protein tyrosine kinases may alter rasGAP activity to induce p 21ras activation in B cells.