Preferential usage of the Fc receptor gamma chain in the T cell antigen receptor complex by gamma/delta T cells localized in epithelia.

zeta and eta chains of the T cell antigen receptor (TCR) complex and the gamma chain of Fc receptors (FcR gamma) constitute a family of proteins important for the expression of, and signal transduction through, these receptors in hematopoietic cells. In zeta-deficient mice, TCR expression was reduced in most T cells. By contrast, CD8 alpha alpha + TCR-gamma/delta + intestinal intraepithelial lymphocytes in these mice expressed a normal level of TCR. Biochemical analysis of the TCR complex in these cells from zeta-deficient as well as normal mice revealed the predominant usage of FcR gamma. Furthermore, gamma/delta + T cells in epithelia of the skin and female reproductive organs from zeta-deficient mice also showed relatively high TCR expression, indicating the usage of FcR gamma. These observations demonstrate the preferential usage of FcR gamma by gamma/delta + T cells localized in epithelia of normal mice.

A novel clinical role for angiopoietin-1 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumour associated with asbestos exposure that has only a limited response to conventional therapy; therefore, diagnosing MPM early is very important. We have previously reported that angiopoietin (Ang)-1 was correlated with bleomycin-induced pulmonary fibrosis. Here, we investigated the association of Ang-1 with the development of MPM cells, which originate from mesenchymal cells similar to lung fibroblasts, and demonstrated that Ang-1 stimulated the growth and migration of MPM cells in vitro. We also demonstrated that patients with MPM had significantly higher serum levels of Ang-1 in comparison to a population who had been exposed to asbestos but had not developed MPM. The patients with advanced-stage MPM showed higher levels of Ang-1 than the early-stage MPM patients and the Kaplan-Meier method revealed a significant correlation between serum Ang-1 levels and survival. We propose the possibility that Ang-1 plays an important role in MPM tumour growth and our data suggest that the serum concentration of Ang-1 could be useful as prognostic factor.

All-trans-retinoic acid inhibits tumour growth of malignant pleural mesothelioma in mice.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumour of mesothelial origin associated with asbestos exposure. Because MPM has limited response to conventional chemotherapy and radiotherapy, the prognosis is very poor. Several researchers have reported that cytokines such as interleukin (IL)-6 play an important role in the growth of MPM. Previously, it was reported that all-trans-retinoic acid (ATRA) inhibited the production and function of IL-6 and transforming growth factor (TGF)-beta1 in experiments using lung fibroblasts. We investigated whether ATRA had an inhibitory effect on the cell growth of MPM, the origin of which was mesenchymal cells similar to lung fibroblasts, using a subcutaneous xenograft mouse model. We estimated the tumour growth and performed quantitative measurements of IL-6, TGF-beta1 and platelet-derived growth factor (PDGF) receptor (PDGFR)-beta mRNA levels both of cultured MPM cells and cells grown in mice with or without the administration of ATRA. ATRA significantly inhibited MPM tumour growth. In vitro studies disclosed that the administration of ATRA reduced 1) mRNA levels of TGF-beta1, TGF-beta1 receptors and PDGFR-beta, and 2) TGF-beta1-dependent proliferation and PDGF-BB-dependent migration of MPM cells. These data may provide a rationale to explore the clinical use of ATRA for the treatment of MPM.

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.

Regimen of tacrolimus-based immunosuppression with basiliximab, mycophenolate mofetil, and low-dose steroid reduces acute rejection in kidney transplants.

BACKGROUND: Acute rejection is a major problem in kidney transplantation. To reduce its likelihood, we investigated the efficacy and safety of an immunosuppressive regimen including tacrolimus, basiliximab, mycophenolate mofetil, and low-dose steroids. METHODS: Fifty-seven patients, including 14 pediatric patients, were enrolled in this study. The mean age at the time of transplantation was 33.5 years, and the mean observation period was 8.2 months. The mean trough concentrations of FK at 1, 6, and 12 months posttransplant were 10.2, 6.6, and 6.0 ng/mL, respectively. RESULTS: All recipients survived without graft loss. The cumulative incidence of acute rejection in adults was 2.3% and 8.4% at 6 and 12 months posttransplant, respectively. Of the adverse events, 11 recipients (19.3%) were positive for CMV antigenemia or had CMV infections. Four recipients (7.0%) exhibited mild hyperglycemia. CONCLUSIONS: Our immunosuppressive regimen demonstrated favorable results, reducing the incidence of acute rejection without causing severe adverse events, especially in adults.

Reversibility from delayed hyperacute rejection in ABO-incompatible renal transplantation: histopathological findings of renal allograft biopsy.

ABO-incompatible renal transplantation (ABOIRTx) tend to lead to blood type antibody-mediated rejection, the so-called delayed hyperacute rejection (DHAR), which results in short-term graft loss. To clarify the accurate incidence and prognostic value of DHAR among ABOIRTx, we reviewed biopsy specimens obtained from ABOKTx allografts with abrupt dysfunction during the early period after transplantation. Among 74 ABOIRTx patients, 34 patients displayed allograft dysfunction within 14 days following transplantation. The biopsy specimens were classified based on the Banff schema. The pathological diagnosis of ABO blood type antibody-mediated humoral rejection (ABO-AMHR) was made by the following 3 findings: Specimens with all of above-mentioned findings were categorized as severe ABO-AMHR; those with at least one findings, were categorized as mild ABO-AMHR. All patients were treated with steroid pulse therapy and/or modification of other immunosuppressants. Group 1 consisted of severe ABO-AMHR (n = 6); group 2 consisted of mild ABO-AMHR (n = 5); group 3 consisted of acute cellular rejection (n = 3); group 4 consisted of recovery phase of ATN (n = 11); group 5 consisted of calcineurin inhibitor toxicity (n = 2); and group 6 consisted of normal histology (n = 5). One of 6 patients (16%) in group 1 lost the graft because of DHAR irreversible by antirejection and anticoagulation therapy. However, there has been no clear definition of histpathological criteria for DHAR after ABO-incompatible kidney transplantation. The definition must prognosticate whether the rejection process is reversible.

Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody.

A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.

Structure of KW-2228, a tailored human granulocyte colony-stimulating factor with enhanced biological activity and stability.

KW-2228 is a tailored human granulocyte colony-stimulating factor (hG-CSF) which has more potent granulopoietic activity and is more stable than wild-type hG-CSF. Analysis of the 2.3 A resolution crystal structure of KW-2228 unambiguously revealed a four-alpha-helix bundle motif with up-up-down-down connectivity. The structures of long overhand loops connecting the helices and the N-terminus have been definitively determined. The present analysis has clearly revealed that substituted residues play important roles in fastening a long overhand loop to the N- and C-termini to fix the conformation. This conformation should be responsible for a substantial enhancement of the biological activity and stability.

Structure-activity relationships for mitomycins. Application of the distance and charge analysis method.

Molecular orbital calculations based on coordinates from X-ray analysis have been performed for a set of 24 mitomycins, of which eight compounds have not been isolated so far. To prioritize the order of synthesis of these missing compounds, a new method named DISCA (distance and charge analysis) has been developed. DISCA screens correlations between spatial distribution of charge in molecules and their biological activity. The spatial distribution of charge is represented by several indexes in DISCA. LD50 and ED50 were used as measures of biological activity. DISCA has successfully extracted indexes which have significantly high correlation coefficients. The indexes with the highest correlation coefficient were common to both LD50 and ED50. By use of the correlation functions with high correlation coefficients DISCA has predicted that 9-epi-1a-N-demethylmitomycin D should have the best ED50 and a modest LD50 among the missing mitomycins.

(E)-4-(2-[[3-(indol-5-yl)-1-oxo-2-butenyl]amino]phenoxy)butyric acid derivatives: a new class of steroid 5 alpha-reductase inhibitors in the rat prostate. 1.

A series of (E)-4-(2-[[3-(indol-5-yl)-1-oxo-2-butenyl]amino]phenoxy)butyric acid derivatives was prepared, and the derivatives were demonstrated to be potent inhibitors of steroid 5 alpha-reductase in the rat prostate. The structure-activity relationships were as follows. An alpha-branched alkyl or benzyl substituent of proper size at position 1 of the indole is crucial for optimal enzyme inhibitory activity. N-Methylation of the amide NH resulted in complete loss of activity. Thus, coplanarity of the benzene ring and amide moiety is essential for such activity. Among the compounds prepared, (E)-4-(2-[[3-[1-[bis(4-fluorophenyl)methyl]indol-5-yl]-1-oxo-2- butenyl]-amino]phenoxy)butyric acid (57, KF18678) was one of the most potent compounds (rat prostate 5 alpha-reductase IC50 = 3.3 nM).

Synthesis and antiallergic activity of 11-(aminoalkylidene)-6,11-dihydrodibenz[b,e]oxepin derivatives.

A new series of 11-substituted 6,11-dihydrodibenz[b,e]oxepin-2-carboxylic acid derivatives was synthesized and demonstrated to be orally active antiallergic agents. These compounds are structurally related to 1 (KW-4994), which we had reported previously to be a new antiallergic agent. Most compounds synthesized exhibited potent inhibitory effects on 48-h homologous passive cutaneous anaphylaxis (PCA) in rats and on IgG1-mediated bronchoconstriction in guinea pigs. Additionally, compounds possessing a terminal carboxyl group at the 2-position of the dibenz[b,e]oxepin ring system exhibited inhibitory effects on specific [3H]pyrilamine binding to guinea pig cerebellum histamine H1 receptors, whereas these demonstrated negligible effects on specific [3H]QNB binding to rat striatum muscarinic acetylcholine M1 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced antiallergic activities: (1) a 3-(dimethylamino)propylidene group as the side chain at the 11-position, (2) a terminal carboxyl moiety at the 2-position, and (3) a dibenzoxepin ring system. Among the compounds synthesized, (Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz [b,e]oxepin-2-acetic acid hydrochloride (16) was selected for further evaluation. It had an ED50 value of 0.049 mg/kg po in the PCA test in rats and an ID50 value of 0.030 mg/kg po in inhibiting anaphylactic bronchoconstriction in guinea pigs. Furthermore, it had a Ki value of 16 +/- 0.35 nM for the histamine H1 receptor, while it exhibited negligible CNS side effects up to a dose of 600 mg/kg po. Compound 16 is now under clinical evaluation as KW-4679.

Molecular forms of circulating adrenomedullin in patients with congestive heart failure.

In the biosynthesis of adrenomedullin (AM), an intermediate form, AM(1-52)-glycine-COOH (iAM), is cleaved from proAM and subsequently processed to a biologically active mature form, AM(1-52)-NH2 (mAM), by enzymatic amidation. We recently reported that immunoreactive AM in human plasma consists of mAM and iAM. To clarify the pathophysiological roles of mAM and iAM in heart failure, we established an assay method to specifically detect mAM, and we determined the plasma concentrations of mAM and iAM in 68 patients with congestive heart failure (CHF). The plasma mAM concentrations of the CHF patients classified as being class I or II of New York Heart Association (NYHA) functional classification were significantly greater than those of the 28 healthy controls, and a further increase was noted in the class III or IV patients. Similar increases in plasma iAM were also observed in these patients compared with controls. The increased plasma mAM and iAM in 12 patients with exacerbated CHF were significantly reduced by treatment of their CHF for 7 days. In addition, the plasma concentrations of both mAM and iAM were significantly correlated with pulmonary capillary wedge pressure, pulmonary artery pressure, right atrial pressure, cardiothoracic ratio, heart rate, and the plasma concentrations of atrial and brain natriuretic peptides in the CHF patients. Thus the plasma concentrations of both mAM and iAM were increased progressively in proportion to the severity of CHF. These results suggest that, though the role of iAM remains to be clarified, mAM acts against the further deterioration of heart failure in patients with CHF.

Effect of chromatic aberration on contrast sensitivity in pseudophakic eyes.

OBJECTIVE: To evaluate the effect of chromatic aberrations in pseudophakic eyes with various types of intraocular lenses (IOLs). PATIENTS AND METHODS: The study included 51 eyes of 33 patients who underwent cataract surgery. The eyes were divided into 3 groups according to the material from which their IOL was made: group 1, polymethyl methacrylate; group 2, silicone; and group 3, an acrylate/methacrylate copolymer. Ten normal phakic control eyes (group 4) underwent the same examination. Best-corrected distance visual acuity and contrast sensitivity were measured under white light and monochromatic light with wavelengths of 470 nm, 549 nm, and 630 nm, with the best correction under white light. RESULTS: There were no significant differences in best-corrected visual acuity and contrast sensitivity under the 549-nm monochromatic light in any group. However, under both white multichromatic light and 470- and 630-nm monochromatic light, the mean contrast sensitivity in group 3 tended to be lower, sometimes significantly, than in the other IOL groups. CONCLUSIONS: Our results showed that longitudinal chromatic aberrations of some IOLs may degrade the quality of the retinal image. Attention must be paid to the detailed optical performance of IOL materials to achieve good visual function.

Management of intraaortic balloon entrapment.

Two cases of intraaortic balloon entrapment were presented. In patient 1, urokinase was injected into the gas driver lumen of the intraaortic balloon catheter, then it could be removed. In patient 2, the intraaortic balloon was removed retrogradely through the left axillary artery. If the entrapped intraaortic balloon is encountered, it is effective to try a clot-lysis method initially, followed by a surgical approach through a left axillary artery.

Alteration of substrate affinity of Streptomyces cholesterol oxidase for application to the rate assay of cholesterol in serum.

The Streptomyces cholesterol oxidase (ChoA) can not be adopted for the rate assay, because the Km value of the enzyme for cholesterol is very small. The choA gene was subjected to random mutagenesis in vivo, and a mutant ChoA (designated E-ChoA) that showed altered substrate affinity was obtained by screening. The Km value of E-ChoA was approximately ten times larger than that of the wild type. Unexpectedly, the thermal stability was also improved. The amino acid substitutions of E-ChoA were identified to be the valine to glutamate at position 145, which has been previously identified as one of the thermostable mutations, and the glycine to serine at position 405. The mutational effects on the structure of E-ChoA are discussed on the basis of a three-dimensional model. E-ChoA has been successfully applied to the rate assay of cholesterol in serum.

Secretion and clearance of the mature form of adrenomedullin in humans.

In the biosynthesis of adrenomedullin (AM), glycine-extended AM, an intermediate form (iAM) processed from proAM is converted to AM[1-52]-NH2, the bioactive mature form of AM (mAM), by enzymatic amidation. We earlier showed that both molecular forms of AM circulate in human plasma. In the present study, to investigate the secretion and clearance sites of mAM and iAM in humans, we examined the plasma mAM and iAM concentrations in the femoral artery and vein (FA and FV), the aortic root and coronary sinus (AO and CS), and the pulmonary artery and capillary (PA and PC) of patients with ischemic heart disease. Plasma mAM in FV was significantly (p<0.001) higher than in FA. There also was a significant (p<0.001) step-up in the plasma mAM of the CS as compared to the AO. In contrast, plasma mAM was significantly (p<0.001) reduced in the PC as compared to the PA. However, such differences were not observed in plasma iAM levels. These findings suggest that in humans the vasculature of the lower extremities and the heart produce and secrete mAM and that the lung is a clearance site of circulating mAM.

Production, characterization and protective effect of monoclonal antibodies to Haemophilus paragallinarum serotype A.

Monoclonal antibodies (mAbs) to Haemophilus paragallinarum serotype A were obtained by fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with whole cells of strain 221. Enzyme linked immunosorbent assay with whole cells was used to show that the monoclonal antibodies are specific for serotype A of H. paragallinarum. Four monoclonal antibodies indicated hemagglutination-inhibition (HAI) activity against serotype A; their titers were 10(4)-10(5). By western blotting, two of these monoclonal antibodies reacted with a protein of molecular weight 39,000. Chickens treated with mAbs possessing HAI activity survived without clinical signs of infection. No challenge strain was isolated from these chickens, indicating that four mAbs with HAI activity suppressed growth of the challenge strain in the nasal cavity, whereas mAbs without HAI activity showed no passive protective effect. These results demonstrated that HI antibodies contributed to protection, and strongly suggest that hemagglutinin (HA) antigen, especially the epitopes which were recognized by these mAbs are important for protective immunity in chickens.

An improved hemagglutination test for study of canine parvovirus.

Optimal conditions for hemagglutination (HA) by canine parvovirus (CPV) strains were investigated using several buffers. Porcine erythrocytes often agglutinated spontaneously in phosphate-buffered salt solution, isotonic saline solution or barbitone-complement-fixation buffer. Results were reproducible when borate-buffered saline (BBS) was used as the diluent for antigen, and "virus adjusting diluent" (VAD), containing 0.15 M NaCl and 0.3 M phosphate was used as the diluent for erythrocytes. Highest HA titers were obtained at pH 6.0 using BBS and VAD. Specific HA with CPV was observed not only at 4 degrees C but at 37 degrees C, and erythrocytes from horse, shrew mouse, hamster, cat, sheep and dog, as well as pig and African green monkey were agglutinated by CPV using the improved method.

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine.

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.

Purification of hemagglutinin from Haemophilus paragallinarum using monoclonal antibody.

The purification of hemagglutinin from Haemophilus paragallinarum was attempted using affinity chromatography with monoclonal antibody. The antigen eluted from the affinity column using potassium thiocyanate buffer agglutinated chicken erythrocytes. In immunoblotting of the eluted antigen, a single band with monoclonal antibody was found as well as the crude antigen. When the chickens were immunized with the eluted antigen, they produced the hemagglutination inhibition (HI) antibody, and they showed protection against challenged exposure with H. paragallinarum strain 221. These results indicated that the HA antigen of H. paragallinarum was a protective antigen.