Characterization of ASKP1240, a fully human antibody targeting human CD40 with potent immunosuppressive effects.

Blocking the CD40-CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.

Use of sonographic elastography of the masseter muscles for optimizing massage pressure: a preliminary study.

To examine the stiffness of the masseter muscle using sonographic elastography and to investigate its relationship with the most comfortable massage pressure in the healthy volunteers. In 16 healthy volunteers (10 men and 6 women), the Masseter Stiffness Index (MSI) was measured using EUB-7000 real-time tissue elastography. They underwent massages at three kinds of pressures using the Oral Rehabilitation Robot (WAO-1). A subjective evaluation regarding the comfort of each massage was recorded on the visual analogue scale. Elastography was also performed in two patients with temporomandibular joint dysfunction with the myofascial pain. The mean MSI of the right and left muscles in the healthy volunteers were 0.85 +/- 0.44 and 0.74 +/- 0.35 respectively. There was no significant difference between the right and left MSI in the healthy volunteers. The MSI was related to massage pressure at which the healthy men felt most comfortable. The two temporomandibular disorder patients had a large laterality in the MSI. The MSI was related to the most comfortable massage pressure in the healthy men. The MSI can be one index for determining the massage pressure.

Tumour lines from a spontaneous rat endometrial stromal sarcoma, showing dendritic cell-like and myofibroblastic cell-like phenotypes.

A transplantable tumour (RY) and cell lines (RY-PB and clone RY-B-E3 isolated from RY-PB) were established from a naturally occurring endometrial stromal sarcoma (ESS) found in a 24-month-old female F344 rat. The primary tumour and RY tumours, which had been serially passaged in syngeneic female rats up to the 10th generation, consisted of spindle or round cells arranged in ill-defined bundles or sheets. Neoplastic cells of the primary and RY tumours, as well as cultured cells of RY-PB and RY-B-E3, showed positive reactions to vimentin, ED1/ED2 (both for rat macrophages/histiocytes), OX6 (for dendritic cells expressing rat MHC class II antigens), and lysosomal enzymes such as acid phosphatase and non-specific esterase, in varying degrees. Ultrastructurally, neoplastic cells characteristically had tubulovesicular system-like structures and variously developed lysosomes in the cytoplasm. Neoplastic cells also exhibited immunoexpression to an alpha-smooth muscle actin (alpha-SMA). The addition of transforming growth factor (TGF)-beta1 to RY-PB and RY-B-E3 cultures increased the number of alpha-SMA-positive cells, whilst the positive cell number was decreased by anti-TGF-beta antibody. The RT-PCR method revealed the expression of TGF-beta1 mRNA in the cultured cells. The present study showed that rat ESS-derived cells exhibited dendritic cell-like and myofibroblastic cell-like phenotypes. The histogenesis of ESSs in human beings and rats remains poorly understood, and these tumour lines may therefore become useful tools for further research.

Effect of FK506 eye drops on late and delayed-type responses in ocular allergy models.

BACKGROUND: It is well-known that FK506 strongly inhibits cytokine production by T cells in vitro. However, less evidence is available from in vivo studies of ocular allergy. OBJECTIVE: To study the anti-inflammatory effect of FK506 eye drops on late and delayed-type responses in several animal models of ocular allergy. METHODS: Rats and guinea-pigs were sensitized with egg albumin (EA) in adjuvant and later challenged by topical EA application to their eyes to examine the late response. Biopsy specimens of conjunctiva were stained with haematoxylin-eosin or stained for T cells and eosinophils. In addition, rats, rabbits and guinea-pigs were sensitized with complete Freund's adjuvant and later challenged by injecting purified protein derivatives for the delayed-type response. Bulbar conjunctival oedema and hyperaemia were graded by score in rabbits, and Evans blue (EB) extravasation was measured in rats and guinea-pigs. FK506 (0.01-1%) and steroid (0.1%) eye drops were instilled in the eyes of animals several times, before and after challenge. RESULTS: FK506 eye drops inhibited T cell and eosinophil infiltration in the late response and EB extravasation in the delayed-type response in rats. Also, they inhibited conjunctival oedema, hyperaemia and ocular mucus in the delayed-type response in rabbits. These effects were similar to those of steroid eye drops (betamethasone sodium phosphate, fluorometholone). FK506 eye drops also inhibited inflammatory cell infiltration, the loss of conjunctival epithelium and decrease of goblet cells in the late response as well as EB extravasation in the delayed-type response in guineapigs, a steroid-resistant species. CONCLUSION: FK506 eye drops inhibit late and delayed-type responses in animal models of ocular allergy.

Characterization of newly established tumor lines from a spontaneous malignant schwannoma in F344 rats: nerve growth factor production, growth inhibition by transforming growth factor-beta1, and macrophage-like phenotype expression.

Transplantable tumor (KE) and clone cell (KE-F11) lines were established from a spontaneous malignant schwannoma found in an aged F344 rat. The primary tumor and KE tumors consisted of oval or spindle cells arranged in ill-defined bundles. Cultured KE-F11 cells exhibited polygonal or spindle configurations. Immunohistochemically, neoplastic cells in KE and KE-F11 reacted to vimentin, S-100 protein, neuron-specific enolase, myelin basic protein, and glial fibrillary acidic protein in varying degrees, indicating neurogenic features; occasional cells reacted to alpha-smooth muscle actin. Cells positive for lysosomal enzymes (acid phosphatase and non-specific esterase), and ED1 (rat macrophage specific) were observed in KE-F11, and electron microscopically, cells with many lysosomes were frequently present, indicating expression of macrophage-like phenotypes. Bioassay analysis revealed that KE-F11 cells produced high levels of nerve growth factor. DNA synthesis was inhibited by addition of transforming growth factor-beta1 (TGF-beta1), and Northern blot analysis revealed that expression of c-myc, a cell cycle-related immediate early gene, was depressed by TGF-beta1. Likely, TGF-beta1 is a factor capable of inhibiting cellular growth of Schwann cells. mRNA expression of the low-density lipoprotein receptor-related protein (LRP) was seen in KE-F11 cells by Northern blot analysis, and the level was decreased by lipopolysaccharide (LPS) treatment. LRP may be attributable to regulation of Schwann cell functions. KE-F11 cells seeded on laminin-coated dishes exhibited more extended cytoplasmic projections than on collagen type I-coated dishes. The present study provides evidence that biological properties of malignant schwannoma-derived cells might be affected by exogenous factors such as TGF-beta1, LPS and laminin. These tumor lines may be useful for studies on pathobiological characteristics of Schwann cells.

Correlation of elevated level of blood midkine with poor prognostic factors of human neuroblastomas.

The heparin-binding growth factor midkine (MK) is the product of a retinoic acid-responsive gene, and is implicated in neuronal survival and differentiation, and carcinogenesis. We previously reported that MK mRNA expression is elevated in neuroblastoma specimens at all stages, whereas pleiotrophin, the other member of the MK family, is expressed at high levels in favourable neuroblastomas. As MK is a secretory protein, it can be detected in the blood. Here, we show a significant correlation of the plasma MK level with prognostic factors of neuroblastomas. The plasma MK level was determined in 220 patients with neuroblastomas, and compared with that in children without malignant tumors (n=17, <500 pg ml(-1)). The plasma MK level became significantly elevated with advancing stages (stage 1: 445 pg ml(-1) (median), n=73; stage 2: 589, n=39; stage 3: 864, n=40; stage 4: 1445, n=56; and stage 4S: 2439, n=12). More importantly, a higher MK level was strongly correlated with poor prognostic factors: over 1 year of age (P=0.0299), MYCN amplification (P<0.0001), low TrkA expression (P=0.0005), nonmass screening, sporadic neuroblastomas (P<0.0001), and diploidy/tetraploidy (P=0.0007). Thus, these results demonstrate that the plasma MK level is a good marker for evaluating the progression of neuroblastomas. Moreover, considering the ability of antisense MK oligodeoxyribonucleotide to suppress tumour growth of colorectal carcinoma cells in nude mice, as recently reported, the present study suggests that MK is a possible candidate molecular target for therapy for neuroblastomas.

Participation of different macrophage populations and myofibroblastic cells in chronically developed renal interstitial fibrosis after cisplatin-induced renal injury in rats.

To shed some light on the mechanisms behind renal fibrogenesis, the present study immunohistochemically investigated the participation of different macrophage populations and myofibroblastic cells in rat renal interstitial fibrosis developed chronically after repeated injection of cisplatin (2 mg/kg body weight, once weekly for 7 weeks). During the 19-week recovery period after the final injection, fibrotic lesions progressively developed in the corticomedullary junction, with the greatest level at post-final injection (FPI) week 5, and then the lesions were gradually repaired by PFI week 19, indicative of a healing process. In conformity with the development of fibrotic lesions, the number of myofibroblastic cells reacting with an anti-alpha-smooth muscle actin antibody was increased, with a peak at PFI week 3, and collagens (types I, III, and IV), fibronection, and laminin were excessively accumulated in these areas. Interstitial cells forming the fibrotic lesions showed mitotic activity at the early stages, whereas they disappeared by apoptosis in the healing process. A large number of cells reacting with an antibody of ED1 (for exudate macrophages), ED2 (for resident macrophages), or OX6 (for major histocompatibility complex class II-presenting macrophages and interstitial dendritic cells) had already appeared at PF1 week 1, and then their numbers increased, with a peak at PFI weeks 7, 3, and 9 in ED1-, ED2-, and OX6-positive cells, respectively. Thereafter, the number of ED1- and ED2-positive cells decreased, whereas the number of OX6-positive cells persisted at a high level until PFI week 19. In the healing process, clusters of lymphocytes were present, the development of which might have been related to OX6-positive cells. The present study demonstrated that chronically developing rat renal interstitial fibrosis might be produced by the complicated mechanisms evoked by interactions between different macrophage populations and myofibroblastic cells, because macrophages show heterogeneous functions depending on microenvironmental factors.

Three-dimensional angioarchitecture in transplantable rat fibrosarcomas.

Three-dimensional angioarchitecture in a transplantable fibrosarcoma (SS) in F344 rats was investigated by scanning electron microscopy (SEM) of vascular corrosion casts. Tumours were produced in syngeneic rats by implantation of a tumour fragment from another SS tumour. Viable SS tumours, observed up to post-implantation (PI) week 5, showed a high degree of vascularization, consisting of arterioles and veins, and intertwining capillaries branching from such vessels. The capillaries showed numerous finger-like outgrowths, indicating active sprouting of endothelial cells and contributing to tumour growth. In contrast, SS tumours in rats given a single dose (4 mg/kg/bodyweight) of cisplatin at PI week 1 were reduced in volume, lacking in capillary outgrowths, and of low vascular density. Necrotic areas in SS tumours were surrounded by capillaries with sharp tips and wrinkled surfaces. Thus, the findings indicated differences in three-dimensional angioarchitecture between vigorously developing tumours and (1) tumours degenerating as the result of treatment with an anti-cancer drug, or (2) necrotic tumour tissues. SEM of vascular corrosion casts may prove useful for the evaluation of neovascularization in mesenchymal tumours.

Differential effects of transforming growth factor-beta1, a fibrogenic factor, on macrophage-like cells (HS-P) and myofibroblastic cells (MT-9) in vitro.

Transforming growth factor-beta1 (TGF-beta1) produced by infiltrating macrophages plays a role in fibrotic disorders through the induction of myofibroblasts. To explore possible mechanisms by which TGF-beta1 may act in this context, we investigated effects of TGF-beta1 on macrophage-like (HS-P) and myofibroblastic (MT-9) cells, two novel cell lines developed by us. Immunocytochemically, the addition of TGF-beta1 (0, 0.1, 0.5, and 1.0 ng/ml) dose-dependently suppressed the expressions of antigens recognized by macrophage/histiocyte-specific antibodies (ED1 and ED2) in HS-P cells, whereas the addition concomitantly increased the number of anti-alpha-smooth muscle actin antibody-positive myofibroblastic cells, suggesting a possible phenotypical modulation of macrophages into myofibroblasts in the fibrotic lesions. By contrast, MT-9 cells did not show such immunophenotypical changes following TGF-beta1 addition. DNA synthesis, measured by tritiated thymidine-incorporation, was inhibited in a dose-dependent manner in MT-9 cells by TGF-beta1 addition (0, 0.1, 0.2, 0.5, 1.0, 5, and 10 ng/ml), but that in HS-P cells was unchanged. Northern blot analysis revealed that expressions of cell cycle-related early genes, c-jun and c-myc, were increased in HS-P cells after TGF-beta1 (1 ng/ml) addition, with c-jun showing peak expression prior to c-myc. By contrast, the peak expressions of c-jun and c-myc were delayed in TGF-beta1 (1 ng/ml)-added MT-9 cells, and their levels were less in MT-9 cells than in HS-P cells. Furthermore, TGF-beta1 (1 and 10 ng/ml) induced DNA laddering in MT-9 cells, but did not in HS-P cells. Based on these findings, it was speculated that TGF-beta1 could have induced G1 arrest in cell cycle and apoptosis in MT-9 cells. The present study showed that there were significant differences in the effects of TGF-beta1 between macrophage-like HS-P cells and myofibroblastic MT-9 cells, presumably depending on divergent susceptibilities to TGF-beta1 between both cell types. Because such cell types are key cells in the fibrogenesis, HS-P and MT-9 might be useful models for investigating the pathogenesis of fibrosis in vitro.

Biorecognizable HPMA copolymer-drug conjugates for colon-specific delivery of 9-aminocamptothecin.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for colon-specific delivery of 9-aminocamptothecin (9-AC) were designed. They hold 9-AC bound via spacers containing amino acid residues and aromatic azo bonds. In vitro release profiles of 9-AC from HPMA copolymer conjugates were evaluated under artificial conditions that simulated large intestinal azoreductase and peptidase activities. The studies indicated that the azo bond was reduced first, followed by the release of unmodified 9-AC from the 9-AC containing fragment by peptidases. Release profiles depended on the chemical structure of the peptide part of the spacer. Conjugates containing leucylalanine showed high colon-specific release of 9-AC when compared to alanine containing conjugates. It appears that the studied conjugates are suitable as colon-specific drug delivery systems.

Effects of lipopolysaccharide on a macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

Lipopolysaccharide (LPS) is a major modulator of macrophage functions. To characterize a newly established rat histiocytic sarcoma-derived cell line (HS-P), immunophenotypic changes and cellular growth responses of HS-P cells exposed to LPS were investigated and compared with those of MT-9 cells isolated from a rat malignant fibrous histiocytoma. MT-9 cells have somewhat histiocytic features, because occasional cells react to rat macrophage-specific antibodies. Addition of LPS to cultured HS-P cells increased the numbers of cells immunopositive to ED1 (rat macrophage-specific antibody) and ED2 (rat histiocyte-specific antibody) and stimulated the phagocytosis of latex beads, whereas LPS-treated MT-9 cells did not show such immunophenotypic changes. LPS-treated HS-P cells showed enhanced immunolabelling of alpha-smooth muscle actin, suggesting a possible modulation of macrophages towards myofibroblastic cells. To evaluate cellular growth after the addition of LPS or fetal bovine serum, DNA synthesis was examined by measuring tritiated thymidine incorporation, and the mRNA expression of c- jun and c- myc (immediate early genes in the cell cycle) was examined by Northern blot analysis. In HS-P cells, the addition of serum greatly increased DNA synthesis and induced high expression of c- jun and c- myc; in contrast, LPS markedly depressed DNA synthesis and reduced the expression of c- jun and c- myc. HS-P cells were more sensitive than MT-9 cells to the growth-promoting effect of serum and the growth-inhibiting effect of LPS. The study demonstrated that HS-P cells are highly LPS-responsive, indicating that they would be useful for studies of macrophage functions.

Immunohistochemical analysis of macrophages and myofibroblasts appearing in hepatic and renal fibrosis of dogs.

Some peptide growth factors produced by macrophages play a role in fibrosis following tissue injury, through the induction of myofibroblasts. In the present study, the appearance of macrophages and myofibroblast development in hepatic and renal fibrosis was determined by immunohistochemical analysis of tissue from 15 dogs. The hepatic and renal fibrosis was classified as grade I, II or III, depending on the extent (percentage) of fibrotic areas per unit area measured by morphometry with Azan-stained sections. The presence of alpha-smooth muscle actin-immunolabelled myofibroblasts was directly related to advancing grade of both hepatic and renal fibrosis. Lysozyme-immunolabelled macrophages also increased in number with increasing grade of hepatic and renal fibrosis. These findings indicate that myofibroblasts and lysozyme-positive macrophages may contribute to progressive fibrosis in canine liver and kidney disease. Interestingly, the number of macrophages recognized by AM-3K, a newly generated monoclonal antibody capable of labelling exuded macrophages and resident tissue macrophages in dogs, fell significantly in grades II and III of renal fibrosis. By contrast, in hepatic fibrosis there were no marked differences in the number of AM-3K-positive macrophages between grades. These findings suggest that there are functional differences between lysozyme- and AM-3K-positive macrophages.

Haptotactic migration induced by midkine. Involvement of protein-tyrosine phosphatase zeta. Mitogen-activated protein kinase, and phosphatidylinositol 3-kinase.

Midkine, a heparin-binding growth factor, plays a critical role in cell migration causing suppression of neointima formation in midkine-deficient mice. Here we have determined the molecules essential for midkine-induced migration. Midkine induced haptotaxis of osteoblast-like cells, which was abrogated by the soluble form of midkine or pleiotrophin, a midkine-homologous protein. Chondroitin sulfate B, E, chondroitinase ABC, B, and orthovanadate, an inhibitor of protein-tyrosine phosphatase, suppressed the migration. Supporting these data, the cells examined expressed PTPzeta, a receptor-type protein-tyrosine phosphatase that exhibits high affinity to both midkine and pleiotrophin and harbors chondroitin sulfate chains. Furthermore, strong synergism between midkine and platelet-derived growth factor in migration was detected. The use of specific inhibitors demonstrated that mitogen-activated protein (MAP) kinase and protein-tyrosine phosphatase were involved in midkine-induced haptotaxis but not PDGF-induced chemotaxis, whereas phosphatidylinositol 3 (PI3)-kinase and protein kinase C were involved in both functions. Midkine activated both PI3-kinase and MAP kinases, the latter activation was blocked by a PI3-kinase inhibitor. Midkine further recruited PTPzeta and PI3-kinase. These results indicate that PTPzeta and concerted signaling involving PI3-kinase and MAP kinase are required for midkine-induced migration and demonstrate for the first time the synergism between midkine and platelet-derived growth factor in cell migration.

Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro.

The objective of this study was to investigate the therapeutic potential of FK506 and other immunosuppressive agents for the treatment of rheumatoid arthritis (RA), focusing on the effects on in vitro IL-6 production and IL-6-mediated immune response. We employed an in vitro model producing IL-6 via T cell activation in human PBMC, based on the hypothesis that T cells play a central role in the pathogenesis of RA. FK506 potently inhibited IL-6 production from PBMC stimulated with anti-CD3 and anti-CD28 monoclonal antibody (anti-CD3/CD28). Cyclosporin A (CsA) also inhibited the anti-CD3/CD28 induced IL-6 production but was about 100 times less potent than FK506. Dexamethasone (DEX) inhibited both anti-CD3/CD28 and LPS induced IL-6 production at almost the same concentration. Methotrexate (MTX) did not affect cytokine production. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance IgM production in SKW6.4 cells. The effects of anti-CD3/CD28 stimulated culture supernatants in the presence of agents on IgM production in SKW6.4 cells were investigated. FK506 and CsA led to suppression of IgM production induced by culture supernatants probably via inhibition of IgM inducible cytokine production from PBMC. DEX profoundly enhanced IgM production, although IL-6 production from PBMC was strongly inhibited by the agent. MTX decreased IgM production although it has no inhibitory effect on IL-6 production. The present study suggests that FK506 is the most effective among the four agents for the suppression of IL-6 production and IL-6-mediated autoantibody production in T cell activation related autoimmune diseases such as RA.

Blood flow in and around the masseter muscle: normal and pathologic features demonstrated by color Doppler sonography.

OBJECTIVE: The purpose of this study was to clarify the normal findings of arteries in and around the masseter muscle and to present their pathologic changes with the use of color Doppler sonography. STUDY DESIGN: The vascular appearances were examined for the 4 main arteries feeding the masseter muscle in healthy volunteers (n = 38) and patients with inflammation (n = 5) and intramuscular hemangioma (n = 3). The features of these arteries were investigated together with the flow diameter, flow velocities, and arterial resistances. The symmetry indices were also calculated to assess the pathologic changes. RESULTS: The detection rates of the branch from the transverse facial artery, the masseter artery, and the branch from the maxillary or external carotid artery were 98.7%, 21.1%, and 84.2% in healthy volunteers, respectively. The facial artery that feeds the muscle from the inferior part represented 2 patterns according to anatomic variant: the masseteric branch (22.4%) and the main trunk itself (77.6%). The means of the flow diameter, maximum and minimum velocities, resistive index, and pulsatility index in healthy subjects were 1.8 mm, 24.6 cm/s, 5.1 cm/s, 0.80, and 2.51, respectively. In most of the patients with symptoms, the symmetry indices of all measurement values increased in comparison with those of healthy volunteers. CONCLUSION: Color Doppler sonography is useful in describing the arteries in and around the masseter muscle and has the potential of being used to depict the pathologic changes.

Malignant fibrous histiocytoma (MFH) cells and macrophages/histiocytes have a common antigen recognized by a monoclonal antibody risen against a rat MFH-derived cloned cell line.

A monoclonal antibody (B9) was generated by using a rat malignant fibrous histiocytoma (MFH)-derived cloned cell line (MT-8) as the immunogen. Immunohistochemically, B9 reacted specifically with a cytoplasmic antigen of MT-8 cells. Furthermore, B9 immunolabeled another MFH-derived cloned cells (MT-9) and histiocytic sarcoma cells, as well as macrophages/histiocytes in normal and diseased tissues of rats. These findings suggest the presence of a common antigen recognized by B9 between MFH cells and macrophages/histiocytes. This suggests that MFH cells may express histiocytic nature.

Design of nanoparticles composed of graft copolymers for oral peptide delivery.

The development of a dosage form that improves the absorption of peptide and protein drugs via the gastrointestinal tract is one of the greatest challenges in the pharmaceutical field. Many researchers have taken up the challenge, using approaches including mucoadhesive drug delivery, colon delivery, particulate drug delivery such as nanoparticles, microcapsules, liposomes, emulsions, micelles, and so on. The objective of this article is to provide the reader with outlines of novel nanoparticle technologies for oral peptide delivery based on polymer chemistry. The physicochemical properties of nanoparticles and their behavior on exposure to physiological media are greatly dominated by their chemical structures and surface characteristics. We will especially focus on the design of nanoparticles composed of novel graft copolymers having a hydrophobic backbone and hydrophilic branches as drug carriers.

An increase in circulating mast cell colony-forming cells in asthma.

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

Tetrahydrobiopterin attenuates modulation of platelet 12-lipoxygenase and cyclooxygenase activities by nitric oxide.

Endothelial cells secrete large amounts of 5,6,7,8-tetrahydrobiopterin (BH(4)) in septic conditions. BH(4) is a cofactor for nitric oxide (NO) synthase and an essential regulator of its activity. We recently showed that NO can be a modulator of both platelet 12-lipoxygenase and cyclooxygenase activities. In the present study, we investigated the effect of BH(4) on the activities of 12-lipoxygenase and cyclooxygenase in rabbit platelets. The influence of BH(4) on NO-induced modulation of these enzyme activities was investigated. Exogenous BH(4) did not affect platelet 12-lipoxygenase and cyclooxygenase activities. The modulatory effects of NO on the two enzymatic pathways were reversed by addition of BH(4) but not by reduced glutathione. These results suggest that exogenous BH(4) is not essential for NO synthase activity of platelets, but that it is an important regulator of the action of NO released from other sources on platelet 12-lipoxygenase and cyclooxygenase activities.

Macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

With future exploration of macrophage properties in mind, we established a novel cell line (HS-P) from a transplantable histiocytic sarcoma, derived originally from a tumour in an aged F344 rat. HS-P was subjected to 70 serial passages, in which the mean doubling time was 15.7 h. The cells, which were round, oval or polygonal in shape, were arranged in a compact sheet. They reacted to varying degrees for lysosomal enzymes (acid phosphatase and non-specific esterase) and with the following antibodies: ED1/ED2 (rat macrophage/histiocyte-specific), OX6 (rat MHC class II-specific), lysozyme antibody and alpha1-antichymotrypsin antibody. Electron microscopically, HS-P cells showed lysosomes and prominent cell projections. These findings indicated that the cultured cells were macrophage-like. Syngeneic rats inoculated subcutaneously or intraperitoneally with HS-P cells invariably developed sarcomatous tumours consisting of monomorphic mononuclear cells, which exhibited cytochemical properties similar to those of cultured HS-P cells. Bioassay and reverse transcription-polymerase chain reaction methods revealed that tumour necrosis factor-alpha increased on addition of lipopolysaccharide (LPS), indicating that HS-P cells remained LPS-responsive. HS-P cells may prove to be a useful tool for in-vitro studies of macrophage function.