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The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A

IL-27 is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4(+) T cells by IL-27, enhanced by TGF-β, and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient-deprivation cell death without releasing high-mobility group box-1. This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Interestingly, Ch25h expressing CD4(+) T cells that received IL-27 and TGF-β signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27 treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worsened outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

Gene expressions of acclimatized and non-acclimatized diapausing larvae were examined in Chilo suppressalis using a subtraction technique. A gene encoding a methionine-rich storage protein, CsSP1, was cloned and its complete cDNA sequence was determined. Potentially, CsSP1 encoded a 758-amino acid protein, with a calculated molecular weight of 88.8 kDa. The expression level of CsSP1 was higher in nondiapausing larvae than in diapausing ones. The CsSP1 expression was up-regulated in diapausing larvae when the temperature of cold acclimation was shifted to 5 degrees C. The up-regulated level was maintained at 40 days after incubation at 5 degrees C. In nondiapausing larvae, CsSP1 expression was down-regulated when the temperature was below developmental zero. Involvement of CsSP1 in diapause, cold tolerance acquisition and postdiapause development in C. suppressalis is discussed.

Pharmacokinetic and metabolic screening plays an important role in the optimization of a lead compound in drug discovery. Since those screening methods are time-consuming and labor intensive, in silico models would be effective to select compounds and guide derivatization prior to the screening. We investigated in silico models for permeability in Caco-2 cells, brain distribution and cytochrome P450 (CYP) inhibition using molecular weight, lipophilicity (clog D(7.4)), polar surface area (PSA), and number of rotatable bonds (RB). A variety of test compounds was selected from different Caco-2 assay projects. The permeability determined exhibited a good correlation with a combination of PSA and clog D(7.4) rather than with PSA alone. In the brain distribution, PSA, in addition to lipophilicity, was one of the determinant parameters, and compounds were significantly distributed to the brain in rats with the decrease in the PSA value. When this approach was adapted to CYP1A2 inhibition in the fluorometric assay, the inhibitory potential for two plane core structures was successfully predicted by utilizing number of RB, PSA, and clog D(7.4). In particular, an increase in the number of RB weakened the inhibitory potential due to a loss of the plane structures. These results suggest that the PSA and RB are key parameters to design chemical structures in terms of the improvement of both membrane permeability in the brain and gastrointestine and CYP1A2 inhibition, respectively.

Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guaçatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.

Background: A study was performed to compare the effects of immunization with ragweed pollen (RW) in two different adjuvants on the characteristics of a previously described model of experimental immune-mediated blepharoconjunctivitis (EC) in rats. Methods: Lewis or Brown Norway (BN) rats were immunized with 100 µg of RW in emulsion with aluminum hydroxide [Al(OH)3] or complete Freund’s adjuvant (CFA). Three weeks later, the animals were challenged with eye drops containing RW in PBS. Twenty-four hours after topical challenge, eyes, blood, and lymph nodes were obtained for histology, measurement of antigen-specific antibodies, and proliferation or cytokine assays, respectively. In addition to active immunization, recipients of RW-primed lymph node cells were challenged and evaluated as above. Results: RW in both adjuvants induced infiltration with predominantly mononuclear cells in Lewis rats and eosinophils in BN rats. As well as active immunization, eosinophils were detected only in BN rats by adoptive transfer of cells. Lymphocyte proliferative responses to RW were high in immunized Lewis rats when CFA was used as an adjuvant. In contrast, proliferative responses in BN rats were higher when Al(OH)3 was used. RW-specific IgE was detected only in BN rats. There were no significant differences in RW-specific IgG1/ IgG2a ratio among the four groups. Lewis rats had higher level of RW-specific interferon-γ in the culture supernatant. Conclusions: The characteristics of EC are different in Lewis and BN rats, dependent on the genetic background of the rat strains. The response to RW was similar to other previously used antigens, such as ovalbumin.

· Background: Fischer rats were less susceptible to experimental autoimmune uveoretinits (EAU) than Lewis rats, although both strains have the same MHC molecules. The purpose of this study was to compare the susceptibility of experimental allergic/immune-mediated blepharoconjunctivitis (EAC) between these two strains. · Methods: Male Lewis and Fischer rats were immunized with either ovalbumin (OVA) or OVA peptide (OVA323–339) in complete Freund’s adjuvant (CFA). Three weeks later, they were challenged with OVA by eye drops. Twenty-four hours later, after clinical evaluation, they were killed and eyes, blood and lymph nodes were harvested for histology, antibody titer and proliferation assay, cytokine production or flow cytometric analysis, respectively. · Results: Fischer rats developed mild EAC compared with Lewis rats. Cellular proliferative responses, IFN-γ production of culture supernatant and serum IgG specific for OVA were basically the same between the two strains. The same OVA peptides were selected as immunodominant. Flow cytometric analysis demonstrated the same cellular profile of lymph node cells in the two strains. · Conclusion: EAC in Fischer rats was milder than that in Lewis rats, although no apparent differences in immunological parameters between these two strains were detected. These data suggest that factors unrelated to immunological parameters may depend on the susceptibility of EAC.

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils.

Previously, we found the first congenital deficiency of histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis. To elucidate the genetic basis of this deficiency, we first performed Southern blot analysis and found no gross deletion or insertion in the proband's HRG gene. We then examined the nucleotide sequences of all seven exons of the proband's HRG gene. A single nucleotide substitution, G to A at nucleotide position 429, which mutates Gly85 to Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22 amplified clones. This mutation generates a unique Taq I site. Exon 3 was amplified from the proband, her family members, and 50 unrelated normal Japanese individuals, and Taq I fragmentation was examined. Fragmentation of exon 3 was observed in one allele of the genes from the proband and the family members who also have decreased plasma levels of HRG. Fifty unrelated normal Japanese individuals had a normal HRG gene, indicating that the G to A mutation is not a common polymorphism. To elucidate the identified mutation as a cause for the secretion defect of HRG in the proband's plasma, we constructed and transiently expressed the recombinant Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK) cells, and examined an intracellular event of the mutant protein. The results showed that only about 20% of the Tokushima-type HRG was secreted into the culture medium, and intracellular degradation of the mutant was observed. Thus, the present study strongly suggests that the HRG deficiency is caused by intracellular degradation of the Gly85 to Glu mutant of HRG in the proband.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

The untranslatable, RNA polymerase II-dependent gene (dutA) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA, requires protein kinase A for its expression.

Fusobacterium nucleatum-derived components were studied for their effect on the ability of accessory pulpal cells to induce T-lymphocyte proliferation. In initial experiments, spleen cells were used to establish concentration ranges at which testing of the effect of the bacterial components would be suitable. At low concentrations, bacterial substances induced a stimulatory effect on the proliferation rate of spleen cells, but not when purified T-lymphocytes were incubated in the presence of concanavalin A and pulpal accessory cells. At increasing concentrations, a dose-dependent reduction was observed. Pretreatment with the bacterial products for 4 h gave a stimulatory effect in the absence of concanavalin A for both spleen cells and T-lymphocytes. Removal of low-molecular weight components (

We examined the role of CD4+ T-cells in peritoneal exudate cells (PECs) during the course of acute murine cytomegalovirus (MCMV) infection in two strains of mice. Cell counts of PECs and cytofluorometric analysis showed that C57BL/6, a resistant strain, had more CD4+ T-cells than BALB/c, a susceptible strain, after intraperitoneal infection of 3 x 10(3) PFU of the Smith strain of MCMV, though both strains had an equivalent number of CD8+ T-cells. CD4+ T-cells of both strains expressed mRNA of IFN-gamma, IL-2, and IL-4 on days 5 and 7 after infection, with much higher expression of these cytokines in C57BL/6 than in BALB/c. At the same time point after infection, macrophages were shown to express mRNA of IL-1 alpha and TNF-alpha with higher expression of IL-1 alpha in C57BL/6 than in BALB/c. Production of nitric oxide, recently shown to be one of the antiviral effector mechanisms of macrophages, by macrophages of both strains was examined showing more production of nitric oxide on day 7 after infection in C57BL/6 than in BALB/c. From these findings, we suggest the possibility that CD4+ T-cells contribute to the protection against MCMV infection via the secretion of cytokines and the resultant activation of macrophages to produce nitric oxide.

A variety of mechanisms can be proposed to explain the potential effects of silicone and silicone by-products on the immune response. In this paper, we discuss information on the chemistry of silicon and silicone gels/elastomers, and the manufacture of silicone breast implants as they pertain to the bioreactivity of silicone. Moreover, with reference to silicone-mediated human adjuvant disease, an overview of experimental adjuvant-induced arthritis is presented; comparisons with graft-versus-host disease and chemically induced autoimmunity then follow. Particular attention is paid to similarities in the characteristics of silicone and classic lipid adjuvants. For example, macrophage activation is presumed to be a central event in silicone-induced autoimmunity. Since those genes uniquely expressed in macrophages activated by plastic adherence are similar to those induced by lipopolysaccharide, adherence to silicone rubber may initiate an inflammatory response by the same mechanism. Macrophage effects would also include the erosion of implants through the generation of oxidants and localized pH changes. The degradation products of silicone are also implicated in the adjuvant effects of silicone implants. There is evidence to suggest that oxidants produced by inflammatory cells preferentially inactivate CD8+ suppressor T cells. This could then lead to an inflammatory state, perhaps through oxidant-induced transcription factors such as NF-kB, resulting in a long-term pro-oxidant imbalance that manifests itself as a breakdown in immunological self-tolerance. The authors hypothesize that autoreactivity following oxidant stress evolved to enhance inflammatory repair mechanisms after tissue, cell or molecular damage by oxidants.

Dietary fish oils rich in 20:5(5,8,11,14,17) and 22:6(4,7,10, 13,16,19) are known to replace arachidonic acid [20: 4(5,8,11,14)] and to improve the immunopathology of New Zealand mice. However, in humans, similar dietary strategics may be impractical because of the high levels of fish oils required. In contrast, we believe that beneficial effects in humans may be attainable using new exotic fatty acids. Toward this end, we have focused on 5,11,14-eicosatrienoic acid [5,11,14-ETA, 20:3(5,11,14)]. This fatty acid is structurally analogous to 20:4(5,8,11,14) but lacks the delta-8 double bond essential for conversion to eicosanoids. To examine our hypothesis, diets containing the oil of Platycladus orientalis containing 3% 5,11,14-ETA, a matched control oil, fish oil, or safflower oil were fed to NZB mice. There was a dramatic delay in both the onset and the titer of direct Coombs' tests in mice fed P. orientalis oil. These were directly reflected by the abundance of 5,11,14-ETA in serum lipids. Most striking was the accumulation of 5,11,14-ETA in serum and tissue phospholipids. Though constituting only 3% of dietary fatty acids, 5,11,14-ETA was the most abundant long chain polyunsaturated fatty acid in the serum phospholipids, suggesting that it very successfully competed with 20:4 as a constituent of membrane lipids. 5,11,14-ETA was incorporated into all tissue phospholipids examined except brain phosphatidyl inositol. Among tissues, liver showed the highest incorporation of 5,11,14-ETA into phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI), yet spleen PE had a higher quantity of ETA than other tissues. Lesser arachidonate in spleen PS, heart PC, and heart PI showed the evidence of replacement by 5,11,14-ETA. The data presented illustrates how new nutrition can modify autoimmune responses and emphasizes the need for further studies based on new nutritional strategies.

We have previously established H-2bm12-restricted autoreactive T-cell clones from NZB.H-2bm12 mice which induce in-vitro production of IgG anti-dsDNA antibodies by syngeneic B cells. However, the mechanism underlying the activation of autoreactive T cells is not clear. We have taken advantage of the existence of L cells which were co-transfected with the A alpha b gene and independent A beta genes comprising all the permutations of amino acid residues distinguishing A beta b from A beta bm12. Using this panel of L cells expressing recombinant I-A molecules, 6/6 autoreactive T-cell lines responded significantly to the FT7.2 L cell transfectant expressing wild-type I-Abm12 as well as control APC from NZB.H-2bm12 or B6.C-H-2bm12 mice, but not to the other recombinant L-cell transfectants or to allogeneic APC. The fixation of APC with paraformaldehyde prior to co-culture led to dramatically diminished reactivity of all the autoreactive cloned T-cell lines tested. Interestingly, the inability of FT7.2 L cells to induce T-cell activation after paraformaldehyde fixation could be reconstituted by the addition of B/monocyte cells, but not T cells, from NZB.H-2bm12, NZB.H-2b or NZB(H-2d) mice and to a significantly lesser extent, from C57BL/6 (H-2b) and BALB/c (H-2d) mice. Conversely, when treated with either mitomycin C or cycloheximide, before incubation with autoreactive T cells and fixed FT7.2 cells, the ability of spleen cells from NZB.H-2b mice to reconstitute reactivity was reduced. Controls for these observations included KLH-specific T-cell clones from NZB.H-2bm12 mice, the T-cell hybridoma H66.3.6.54 specific for beef insulin and restricted to I-Ab, the IBM026 hybridoma specific for OVA and restricted to I-Abm12, and the TH2.2 hybridoma, and I-Ab antigen-presenting cell line.

We have previously demonstrated the influence of the I-Abm12 gene mutation on the appearance of IgG anti-dsDNA antibodies when placed on an NZB genetic background. To further enhance our understanding of the interaction of the bm12 mutation on disease expression, lethally irradiated NZB.H-2bm12/b F1 mice were reconstituted with T-cell-depleted bone marrow cells from 3- to 6-week-old donors of four different congenic strains, NZB.H-2bm12, NZB.H-2b, B6.C-H-2bm12 and C57BL/6 (H-2b) mice. All animals when then serially followed for the appearance of IgM and IgG anti-ss and dsDNA antibodies. Significant alterations of T-cell subsets, high levels of IgG anti-dsDNA antibodies and proteinuria were found only in recipient NZB.H-2bm12/b F1 mice that were reconstituted with T-cell-depleted NZB.H-2bm12 bone marrow cells. Such activity was not found in F1 mice engrafted and fully reconstituted with NZB.H-2b, B6.C-H-2bm12 or C57BL/6 (H-2b) T-cell depleted bone marrow cells. Additionally, to evaluate the importance of the NZB background (non-H-2) genes, we transferred T-cell-depleted bone marrow cells from NZB.H-2bm12 mice into H-2 compatible B6.C-H-2bm12 mice and vice versa. Without NZB background genes, an increase in CD4- CD8- T cells, IgG anti-dsDNA, splenomegaly, and proteinuria were not observed. These data suggest that the H-2bm12 gene and the NZB background genes in bone marrow-derived cells are both necessary for the altered expression of T-cell subsets and anti-DNA production.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Central noradrenergic neurones have a role in alertness, analgesia, and thermoregulation; these neurones are also involved in the mechanism of anaesthesia. Locus coeruleus neurones innervate various central nervous regions including the cerebral cortex and hippocampus, and are responsible for wakefulness and analgesia. We hypothesized that these neurones are also involved in both activation of the γ-aminobutyric acid type A (GABAA) receptor and inhibition of N-methyl- d -aspartate (NMDA) receptor-mediated anaesthesia. Methods Forty-seven male rats were used to compare duration of anaesthesia before and 10 days after noradrenergic neurone depletion after intraperitoneal (i.p.) administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 0, 5, and 50 mg kg−1). The animals received i.p. thiopental (GABA anaesthetic, 45 mg kg−1) or ketamine (NMDA anaesthetic, 100 mg kg−1). We also tested the effects of coerulean noradrenergic neurone depletion on i.p. ketamine analgesia (15 mg kg−1) using the hot-plate test in a further 21 male rats. At the end of each experiment, norepinephrine contents in the cerebral cortex and hippocampus were measured. Results I.P. DSP-4 5 and 50 mg kg−1 significantly decreased ketamine anaesthesia duration by 12.7% and 22.4%, increased thiopental anaesthesia duration by 10.8% and 24.5%, and reduced ketamine-increased hot-plate latency by 55.2% and 68.1%, respectively. In addition, i.p. DSP-4 5 and 50 mg kg−1 significantly reduced norepinephrine contents in coerulean brain regions by ∼20% and ∼75%, respectively. There were significant correlations between norepinephrine contents in coerulean brain regions and anaesthesia durations and ketamine analgesia. Conclusions The present data indicate that coerulean noradrenergic neurones may be responsible for both GABA- and NMDA-mediated anaesthetic actions.

Interleukin (IL)-27 and WSX-1, the receptor α-specific subunit, have been shown to play important roles in initiating Th1 responses and in inducing immune modulation, and the immunosuppressive effect of IL-27 appears to be exerted via suppression of IL-10 and IL-17, which may participate in the pathogenesis of human systemic lupus erythematosus (SLE).To examine the significance of IL-27/WSX-1 signalling in spontaneous skin inflammation of MRL/lpr mice, a model for SLE.The severity and development of skin lesions, dermal inflammatory cells and epidermal-dermal depositions in the skin lesions of MRL/lpr mice with CD2-promoted WSX-1 overexpression (WSX-1 Tg mice) and those with globally disrupted WSX-1 (WSX-1 KO mice) were examined and compared with those of MRL/lpr mice.By 4 months of age, both WSX-1 KO mice and control MRL/lpr mice developed predominantly similar skin inflammation, while WSX-1 Tg mice hardly did so, demonstrating that intensifying IL-27/WSX-1 signalling on T cells prevents the spontaneous skin inflammation. WSX-1 KO mice showed Th2-type skin inflammation as evidenced by the Th2-prone dermal infiltrating cells and an absence of cutaneous Th1-type IgG deposition. Interestingly, there were significant IL-17+ dermal infiltrating cells in both WSX-1 KO and control MRL/lpr mice, which might potentially contribute to the formation of skin inflammation in these mice.These data indicate that IL-27/WSX-1 signalling may play a protective role in the development of SLE-like skin inflammation, and modulating IL-27/WSX-1 signalling might be an interesting therapeutic strategy in the treatment of SLE.

A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman (IS) that induced sperm agglutination of human and boar sperm. The antisperm antibodies interacted with a 50-kD boar sperm protein, determined by immunoblot. The 50-kD protein was extracted with deoxycholate (DOC) and purified by affinity chromatography on concanavalin A column, ion exchange chromatography on CM52 column, affinity chromatography on IS IgG-protein G-agarose, and by preparative electrophoresis with electroelution. The purified 50-kD protein migrated as a single homogeneous band when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Western blot. The production of antibodies against specific sperm proteins may be a cause of immunological infertility.

Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naive and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.

Adenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune (CMI) responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Adenovirus serotype 5 (Ad5) vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing the E.C7 cell line for viral packaging. Its' effectiveness as a potential vaccine platform as compared to the currently utilized Ad5 [E1-]-based platform was assessed in both Ad5 naive and Ad5 immune mice. We employed the HIV-1 Gag gene as the antigenic transgene expressed by the novel vector. Cellular expression of the Gag was confirmed by Western Blot analysis. Dose response studies using three intradermal immunizations of 10(7) to 10(10) virus particles (VP) of each construct revealed that immunization with 10(10)VP resulted in the maximum immunological response. Multiple immunizations of Ad naive BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in higher ELISpot CMI responses as compared to mice immunized with an Ad5 [E1-]-gag vaccine. More importantly, multiple immunizations of Ad5 immune BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in significant increases in ELISpot CMI responses when compared to Ad5 immune mice vaccinated with an Ad5 [E1-]-gag vector. Preliminary studies in three Ad5 immune non-human primates (NHP) demonstrated that vaccination with Ad5 [E1-, E2b-]-gag-induced elevated levels of interferon-gamma and IL-2 secreting lymphocytes as assessed by ELISpot assays. These studies indicate that the novel Ad5 [E1-, E2b-] viral vector can be utilized as a potential vaccine platform to induce elevated CMI responses as compared to current generation Ad5 [E1-] viral vectors even in the presence of pre-existing Ad5 immunity.

Background The antitumor activity of CS-1008, a humanized agonistic anti-human death receptor (DR) 5 antibody, was investigated in preclinical models. Materials and methods Cytotoxicity of CS-1008 was evaluated in a several human tumor cell lines as well as primary human hepatocytes in vitro. To evaluate antitumor efficacy, athymic nude mice were inoculated with human colorectal tumor COLO 205, pancreatic tumor MIA PaCa-2 or non-small-cell lung carcinoma NCI-H2122 and CS-1008 was i.v. administered. The combination effects of CS-1008 with gemcitabine or docetaxel (Taxotere) against MIA PaCa-2 or NCI-H2122 were evaluated in vivo, respectively. Results CS-1008 inhibited the growth of tumor cell lines with DR5 expression, including COLO 205, NCI-H2122, MIA PaCa-2 and renal cell adenocarcinoma ACHN in vitro with antibody cross-linkage. Using COLO 205, apoptosis induction was confirmed by annexin V staining. Weekly administration of CS-1008 resulted in the inhibition of COLO 205 tumor growth as well as MIA PaCa-2 in vivo. CS-1008 in combination with gemcitabine or docetaxel demonstrated enhanced antitumor activity against MIA PaCa-2 or NCI-H2122 cells, respectively. Unlike tumor necrosis factor-related apoptosis-inducing ligand, CS-1008 did not induce cell death in human primary hepatocytes. Conclusion CS-1008 has a selective toxicity toward tumor cells expressing DR5 and the potential for antitumor efficacy in human malignancies.

The influence of topical PUVA on the activity of ornithine decarboxylase (ODC) and its gene expression was investigated in the skin of hairless mouse. After 1 h of application of 0.3% 8-methoxypsoralen, irradiation of 3 J/cm2 of ultraviolet A (UV-A) was administered. The ODC activity markedly increased and peaked at 24 h following UV-A irradiation. The ODC raRNA level analyzed in dot-blot analysis elevated to about 5 times that of control at 24 h after treatment. These results show that the PUVA-induced ODC activity is due in part to an increase in ODC gene expression.

Promyelocytic leukemia (PML) is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis and myeloid cell differentiation. PML is subjected to post-translational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially for myeloid cell differentiation, remains unclear. In this report, we found that four serine residues in the PML C-terminal region are highly phosphorylated in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, but a phosphorylation-deficient PML mutant failed. PML interacted with C/EBP epsilon, a transcription factor essential for granulopoiesis, activated C/EBP epsilon-mediated transcription in concert with p300 and accelerated C/EBP epsilon-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBP epsilon-dependent transcription and accelerating C/EBP epsilon-induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways.

Background: Ischemia reperfusion (I-R) injury of the lung frequently occurs after cardiopulmonary bypass, pulmonary thromboendarterectomy, lung transplantation, and major pulmonary resection with vascular reconstruction. Mild hypothermia ameliorates ischemia reperfusion injury of the brain and the liver. However, the effect of mild hypothermia on I-R injury of the lung has not been investigated. Methods: The lungs of Lewis rats underwent 80 min of ischemia followed by 60 min of reperfusion in an ex vivo perfusion model. The ambient temperature was maintained at either normothermia (38°C, n = 6) or mild hypothermia (35°C, n = 6) during the ischemia and reperfusion. Results: Pulmonary shunt fraction, peak inspiratory pressure, mean pulmonary arterial pressure during reperfusion, and the wet/dry weight ratio of the lung tissue at the end of reperfusion in the mild hypothermia group were significantly (p < 0.05) lower than those in the normothermia group. Total adenine nucleotide, adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate after reperfusion in the mild hypothermia group were significantly (p < 0.05) higher than those in the normothermia group. Conclusion: Mild hypothermia attenuates I-R injury of the lung with maintained levels of intrapulmonary high-energy phosphate compounds after reperfusion, suggesting its beneficial effect on warm lung I-R in clinical settings.

Metabolic screening using liver microsomes of rats and humans is an indispensable tool to optimize a lead structure and to select compounds for in vivo study. Elucidating the relationship between in vitro intrinsic clearance (CL(int, app)) and in vivo clearance (CL(b)) is a prerequisite for screening. We investigated the relationship between CL(int, app) in rat liver microsomes and CL(b) after intravenous administration in rats in eight projects. No relationship between these two parameters was found across all of the projects examined. However, there was a certain relationship in the same core structure of six projects, but not in the other two projects. The poor correlation in the projects was improved by considering serum protein binding or microsomal binding in the estimation of in vitro clearances. Although the binding assay was labor intensive, unlike metabolic screening, the introduction of the equilibrium dialysis method using a 96-well format increased the throughput. Optimization of metabolic stability was conducted on the basis of the structure-metabolic stability relationship (SMR) in one of the projects, showing a good correlation without the binding factors. The replacement of the piperazine with a homopiperazine moiety improved metabolic stability in the rat and human liver microsomes. The compound also showed a desirable in vivo pharmacokinetic profile in rats, suggesting that the SMR study on the confirmed in vitro and in vivo correlation is essential to the optimization.

To examine the effect of LY309887, an inhibitor of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis on murine type collagen-induced arthritis (CIA).CIA was induced by immunization with bovine type II collagen in adjuvant. The expression of folate receptors was examined in dissected synovial tissues and bone marrow cells from arthritic and non-arthritic mice by the semi-quantitative reverse transcription-polymerase chain reaction. LY309887 was administered to CIA mice after the onset of arthritis. Mice were monitored for arthritis index for 21 days. Levels of IgG1 and IgG2a antibodies against bovine type II collagen were measured in sera from CIA mice with or without LY309887 treatment by the enzyme-linked immunosorbent assay. Histologic analyse were also performed in synovial tissues from arthritic joints with or without LY309887 treatment.Levels of mRNA of folate receptor beta (FR-beta) were elevated in arthritic joints from CIA mice, compared with those in nonarthritic joints. The expression of mRNA of FR-beta was dominant in bone marrow cells of CIA mice. The administration of LY309887 suppressed the disease progression of CIA mice as defined by the lower arthritis index, and decreased production of serum IgG1 and IgG2a anti-type II collagen antibody, and the damage to cartilage or bone.The administration of LY309887 was effective on CIA mice. It was suggested that LY309887 might be useful in the treatment of rheumatoid arthritis.

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.

This article summarizes our efforts to use chromosome-based vectors for animal transgenesis, which may have a benefit for overcoming the size constraints of cloned transgenes in conventional techniques. Since the initial trial for introducing naturally occurring human chromosome fragments (hCFs) with large and complex immunogulobulin (Ig) loci into mice we have obtained several lines of trans-chromosomic (Tc) mice with transmittable hCFs. As expected the normal tissue-specific expression of introduced human genes was reproduced in them by inclusion of essential remote regulatory elements. Recent development of 'chromosome cloning' technique that enable construction of human artificial chromosomes (HACs) containing a defined chromosomal region should prevent the introduction of additional genes other than genes of interest and thus enhance the utility of chromosome vector system. Using this technique a panel of HACs harboring inserts ranging in size from 1.5 to 10 Mb from three human chromosomes (hChr2, 7, 22) has been constructed. Tc animals containing the HACs may be valuable not only as a powerful tool for functional genomics but also as an in vivo model to study therapeutic gene delivery by HACs.

We have analysed the neurovirulence of Sabin 1-derived isolates which persisted more than nine years in an immunodeficient patient in the U.S.A. Samples were collected from stool specimens at days 11 (St1), 23 (St2), 48 (St3), 126 (St5), and 200 (St7) after the onset of paralysis. Critical nucleotides associated with the reversion of virulence were examined. All the isolates had the substitutions at nucleotide positions 480 (G to A) in the 5'-non-coding region (NCR), 2438 (A to U) in VP3, 2795 (A to G) in VP1, and 6203 (C to U) in 3D. Serially diluted samples were injected intracerebrally to transgenic mice harbouring the human poliovirus receptor gene. Samples St2, 3, 5 and 7 showed typical virulent characters in transgenic mice, whereas the sample ST1 showed intermediate neurovirulence. It seemed that there were two variant viruses providing for a major (M) and a minor (m) populations. After disappearance of the m-variant, samples obtained at the later stages showed neurovirulence almost equivalent to that of the Mahoney strain. Thus, the Sabin 1 strain evolved towards full neuropathogenicity after long-term replication in humans by accumulating mutations. Therefore, OPV-vaccination of immunodeficient persons should be avoided.

The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage.We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells.These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.

Although there are several limitations, optical recording techniques are superior to multi-electrode mapping methods, as it is possible to record at large number of points in a small area without destroying the tissue and possible to know relative changes of membrane potentials. Optical recording techniques using voltage-sensitive dyes will be more importantly applied in the study of central respiratory control (e.g., mechanisms of respiratory rhythm generation) in the near future.

Inactivation of the retinoblastoma (Rb) tumor suppressor in the mouse induces mid-gestational death accompanied by massive apoptosis in certain tissues. Herein, we analyzed the role of the apoptosis protease-activating factor Apaf-1, an essential component of the apoptosome, in mediating apoptosis in Rb-deficient mice. Analysis of compound mutant embryos lacking Rb and Apaf-1 revealed that Apaf-1 was absolutely required for apoptosis in the central nervous system and lens. In contrast, apoptosis in the peripheral nervous system and skeletal muscles only partly depended on Apaf-1 function. The dependency on Apaf-1 coincided with the requirement documented previously for E2F1 and p53 in the respective tissues. Loss of Apaf-1 specifically suppressed apoptosis but not the proliferation and differentiation defects in Rb-mutant embryos. We also show that the Apaf1+ but not the Rb+ allele is retained in pituitary tumors arising in Rb+/-:Apaf1+/- double heterozygous mice. Our results indicate that Apaf-1 plays a critical role in apoptosis in a subset of tissues and that both E2F1:p53:Apaf-1-dependent and -independent apoptotic pathways operate downstream of Rb.

In tumor-bearing animals we found that the skeletal muscle apoptosis might be involved in muscle wasting. In this study, we investigated changes in the skeletal muscle cell apoptosis regulatory proteins after cyclic plasma-perfusion (CPP).We studied changes in body weight, lean body mass (LBM), apoptotic index (AI) and expression of Bax and Bcl-2 in skeletal muscle in VX2 carcinoma-bearing rabbits.20 days after tumor implantation, LBM had decreased by 5.06+/-1.10%, while the AI had increased to 40.5+/-3.20%. By 40 days, LBM had decreased by 11.0+/-0.81% and the AI was only 0.93+/-0.96%. Bax expression was detected in proportion to the AI, but no Bcl-2 expression was detected in either the experimental or control groups. CPP improved LBM, but did not prevent Bax expression.Skeletal muscle cell apoptosis related to Bax was concluded to be the cause of muscle wasting in VX2 carcinoma-bearing rabbits. CPP appears to reduce muscle wasting and increase LBM, but it did not suppress Bax expression or skeletal muscle cell apoptosis.

DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear.We over-expressed DREF in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF-32.The DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.

1. The effect of long-term treatment of rats with chronic heart failure (CHF) following acute myocardial infarction with trandolapril, an angiotensin I-converting enzyme (ACE) inhibitor, on heat shock-induced Hsp72 and Hsp73 production was examined. 2. Acute myocardial infarction was induced by coronary artery ligation (CAL). The animals with CAL showed symptoms of CHF at the 8th week after the operation. The hearts isolated from animals with CAL at the 2nd and 8th week after surgery were subjected to hyperthermia at 42 degrees C for 15 min followed by 6-h perfusion (hyperthermia/6-h perfusion). 3. In the hearts isolated from the animals at the 2nd week, an approximate 20% decline in the rate pressure product (RPP) was seen after hyperthermia/6-h perfusion, which was similar to that in non-operated controls. In contrast, a significant reduction in the RPP after hyperthermia/6-h perfusion was seen in the hearts of rats with CHF. These hearts did not increase Hsp72 and Hsp73 production after hyperthermia. The decline in RPP was associated with failure in the production of myocardial Hsp72 and Hsp73. 4. When rats with CAL were treated with 3 mg kg(-1) day(-1) trandolapril from the 2nd to 8th week after the operation, the decline in RPP of the failing heart after hyperthermia was similar to that of the sham-operated rats. The induction of myocardial Hsp72 and Hsp73 production of the coronary artery-ligated rats after hyperthermia was reversed by treatment with trandolapril. 5. These findings suggest that the preserved ability to induce Hsp72 and Hsp73 production in the heart with CAL by trandolapril treatment may be attributed to the increased tolerance against heat stress-induced deterioration of myocardial contractile function.

Intestinal mucosa is continuously exposed to mechanical forces. We examined whether pressure loading activates mitogen-activated protein kinase (MAPK) and expression of early immediate genes in intestinal epithelial cells. Pressure was applied to IEC18 cells by helium gas in a culture flask and pressure-induced cell proliferation was examined. The expression of early immediate genes, MAPK activity, and activation of nuclear factor activator protein-1 (AP-1) were also examined. Pressures significantly promoted cell proliferation with peak effect at 80 mm Hg. Pretreatment with either a protein kinase C inhibitor or tyrosine kinase inhibitors, but not calcium chelating agents significantly inhibited cell proliferation promoted by pressure. Early inductions of c-myc and c-fos proteins, increased activity of MAPK, and activation of AP-1 were observed by pressure loading. Our study showed that intestinal mucosal cell proliferation is promoted by mechanical pressure and various intracellular signaling pathways are involved in the process.

We encountered a patient with a mild fever and chest discomfort in whom right pleural effusion and eosinophilia (48%) had developed. Parasite eggs were found in the feces, and metagonimiasis was diagnosed. The pleural fluid contained many Charcot-Leyden crystals, suggesting active eosinophilic inflammation. Metagonimus yokogawai usually occurs as a gastrointestinal parasite and causes only mild gastrointestinal symptoms such as diarrhea. Most cases of metagonimiasis resolve spontaneously. However, the main manifestation of metagonimiasis in our patient was pleural effusion. Some cases of idiopathic eosinophilic pleural effusion of unknown origin resolve spontaneously, as in our patient. Patients who are therefore considered to have idiopathic pleural effusion may include those with transient parasitic infection.

The biotransformation of esonarimod (KE-298) [(+/-)-2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid], a new antirheumatic drug, was investigated in rats. Urinary and biliary excretions within 24 h after oral administration of 5 mg/kg [(14)C]esonarimod accounted for 89 and 10% of the dose, respectively. Initial metabolite analysis by liquid chromatography/electrospray ionization tandem mass spectrometry with negative ion mode, in which a mobile phase of 20 mM ammonium acetate (pH 4.6)/methanol with gradient-elution mode was used, failed to obtain any structural information on most of the metabolites due to poor sensitivity. To overcome this problem, postcolumn addition of 2-(2-methoxyethoxy)ethanol, a powerful signal-enhancing modifier, to the mobile phase was used, allowing pronounced signal enhancement and structural elucidation of urinary and biliary metabolites. The results of metabolite analysis suggested that esonarimod is predominantly biotransformed to a pharmacologically active metabolite, thiol-containing deacetyl-esonarimod (M-I), and subsequently undergoes extensive metabolism, mainly S-methylation followed by the combination of S-oxidation and oxidative conversion of the aromatic methyl group. No disulfide metabolites, such as M-I-cysteine mixed disulfide and M-I-dimer, were found in the excreta. This finding is probably evidence supporting the notion that the reactivity of the thiol moiety of M-I with macromolecules in vivo is extremely lower than that of common thiol-containing drugs.

The proliferation of mouse submandibular gland carcinoma YT-12 cells was stimulated by endothelial cell growth factor (ECGF)/bovine brain-derived acidic fibroblast growth factor (aFGF) and recombinant human aFGF. To determine whether aFGF was capable of modifying salivary gland carcinogenesis, the effect of brain-derived aFGF was examined in vivo. Mice in Groups 1 and 2 were injected with 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received bovine brain-derived aFGF and Group 2 mice received vehicle subcutaneously for 10 weeks. Group 3 and 4 mice received either bovine brain-derived aFGF or vehicle only. Sixteen weeks after the start of the experiment, the incidence of submandibular gland carcinomas in Group 1 was significantly greater than that in Group 2. Immunohistochemical study indicated that ducts in the normal submandibular glands and carcinomas showed positive staining with anti-aFGF antibody. Immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression of aFGF in these tissues. FGF receptor (FGFR)-1 and FGFR-4 were detectable in the mouse submandibular glands and carcinomas. These findings suggest that bovine brain-derived aFGF stimulates the proliferation of submandibular gland carcinoma cells and promotes mouse submandibular gland carcinogenesis.

It has not clearly been elucidated how differently differentiation-inducing drugs act on tumor cells, whether they promote differentiation or apoptosis. To elucidate the mechanisms whether leukemic cells responding to ONO-4007, a lipid A derivative, undergo differentiation or apoptosis, we established two cell clones from a rat myelomonocytic leukemia c-WRT-7/P2 clone which undergoes differentiation followed by apoptosis by ONO-4007-treatment. One of the clones (1D6) showed the features of differentiation, such as phagocytosis when treated with ONO-4007 more than 24 hrs. The other clone (3B1) clearly showed the features of apoptosis, such as DNA ladder formation within 24 hrs after incubation with ONO-4007. We then examined expression of CD14, p21, p38MAPK, JNK/SAPK, and bcl-2, functional p53 statuses and cell cycle in these two clones, and revealed the following: Without treatment with ONO-4007; 1) CD14, p21, and bcl-2 proteins were equally expressed in both clones; 2) wild-type and non-functional mutated-type p53 were present in both clones and the p53 in 3B1 clone was recessive whereas that in 1D6 clone was dominant negative; 3) p38MAPK in 3B1 clone was already phospholyrated whereas that in 1D6 clone was not. After treatment with ONO-4007; 1) neither expressions of CD14 nor that of p21 protein was changed in any of the clones; 2) p38MAPK in 3B1 clone was dephospholyrated at 1 and 2 hrs after treatment whereas that in 1D6 clone was phospholyrated at 4 and 8 hrs after treatment; 3) the expression of bcl-2 protein in 3B1 clone was reduced. These findings suggest that p53 may be one of the key factors in leading these cells to differentiation or apoptosis, and that bcl-2 may suppress the apoptosis.

The anti-metastatic efficacy and safety of a newly-developed matrix metalloproteinase (MMP) inhibitor were examined. MMI-166, a N-sulfonylamino acid derivative, inhibited the enzyme activity of MMP-2, 9, and 14 but not MMP-1, 3 or 7. Daily oral administration of MMI-166 resulted in potent inhibition of metastatic lung colonization of Lewis lung carcinoma injected via the tail vein and liver metastasis of C-1H human colon cancer implanted into the spleen at inhibition levels of 43% and 63%, respectively. Daily administration of MMI-166 also resulted in prolonged survival of mice given intraperitoneal implantation of Ma44 human lung cancer cells. The anti-metastatic activity of MMI-166 was as effective as that of other MMP inhibitors with broad inhibitory spectrum. MMI-166 did not affect in vitro tumor cell growth. Neither body weight losses nor hematotoxicity was observed during long-term treatment, indicating the safety of MMI-166 in mice. These results indicate that the selective MMP inhibitor MMI-166 has therapeutic potential as an anti-metastasis agent.

Clarithromycin (CAM) was synthesized by selective methylation of the C-6-hydroxy group of erythromycin (EM), which had a high affinity to the lung. In the present study, the relationship between the chemical structure of derivatives of O-methyl group for the hydroxy group at 6, 11, 12 and 4"-position of the EM and their affinity to the lung was investigated. When each O-methyl EM derivatives was injected into the external jugular vein in rats, the lung levels were in decreasing order, 6,11,12,4"-OCH36,12-OCH36,11,4"-OCH36,4"-OCH36,11-OCH36-OCH311-OCH3EM. There was not a correlation between the lung affinity and dissociation constant (pKa). It was found that substitution of a O-methyl group for the hydroxy group at each position afforded both the affinity to the lung and the lipophilicity. However, the levels of the increase were inconsistent for each substitution position. In the case of 6 and 12-position afforded higher affinity than that for 11 and 4"-position. A study of the effect of EM derivatives on the [14C]EM and [14C]CAM uptake process into the isolated lung cells was carried. The uptake of [14C]EM was not influenced by EM, CAM and 6,11,12,4"-OCH3. On the other hand, the uptake of [14C]CAM was significantly inhibited in the presence of derivatives which had a O-methyl substitution at position 6, but was not influenced by an O-acethyl substitution. These findings suggest that CAM is transported via carrier-mediated system in the lung cells, which has a selective recognition the O-methyl group at position 6. In addition, the uptake of [14C]CAM was further inhibited by the derivatives with O-methyl group at position 12.

Recent studies revealing that endothelial cells derived from E8.5-E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known. We found that mice deficient in Runx1/AML1 (an alpha subunit of the transcription factor PEBP2/CBF) lack c-Kit+ haematopoietic cell clusters in the dorsal aorta, omphalomesenteric and umbilical arteries, as well as yolk sac vessels. Moreover, endothelial cells sorted from the embryo proper and the yolk sac of AML1-/- embryos are unable to differentiate into haematopoietic cells on OP9 stromal cells, whereas colonies of AML1-/- endothelial cells can be formed in culture. These results strongly suggest that the emergence of haematopoietic cells from endothelial cells represents a major pathway of definitive haematopoiesis and is an event that also occurs in the yolk sac in vivo, as suggested by earlier in vitro experiments.

A retroviral insertional mutation, especially by mouse mammary tumor virus (MMTV), is a major cause of murine mammary tumorigenesis. Prompted by our previous finding that FGF8, an insertionally activated cellular oncogene, is highly expressed in androgen-dependent mouse mammary Shionogi carcinoma cells, we here investigated retroviral integration adjacent to the fgf8 locus in Shionogi carcinoma. In the genomic Southern blots for fgf8 and its 5'-upstream gene npm3, the hybridized fragments were identical to the host DD/Sio mice, the original Shionogi carcinoma 115 tumor, and a pair of cultured Shionogi carcinoma cell lines of SC-3 and SC-4, suggesting that no retroviral integration occurred around either loci. The genomic cloning for the fgf8 locus from SC-3 cells also confirmed no MMTV integration. In addition, npm3, which is usually coactivated with fgf8 by MMTV insertion,was not up-regulated by androgens in SC-3 cells. All these findings led us to conclude that no retroviral insertion was present at the common integration sites adjacent to the fgf8 locus in Shionogi carcinoma although we demonstrated in this study that multiple proviral sequences of MMTV, Moloney murine sarcoma virus and FBJ-murine sarcoma virus are integrated into SC-3 cells in association with their distinct promoter activity in SC-3 cells.

Monocyte/macrophage (m phi) or mononuclear phagocytic system(MPS) play one of the key roles in defense mechanisms in human as well as animals. Informations as to their biological functions and significance in defense mechanisms or immune systems have increased. However, some of the details are still unknown and/or controversial. Prof. Takahashi lectured on some new findings: development of m phi precedes that of monocyte, factors regulating differentiation of m phi, differences between tissue m phi and monocyte/m phi and so on. He also stressed the complexity of the development and differentiation of monocyte/m phi. Prof Yamamoto lectured on a new factor, S19 ribosomal protein, which enhances migration of monocytes and connects natural immunity and acquired immunity. Following these lectures, all participants realized the complexity and also the mystery of defense mechanisms of our bodies.