Epinastine inhibits eosinophil chemotaxis and adhesion molecules in atopic dermatitis.

PURPOSE: To investigate the effects of epinastine on eosinophil chemotaxis and changes in eosinophil adhesion molecules induced by epinastine and three other antiallergic agents, using eosinophils of atopic dermatitis (AD) patients. RESULTS: Epinastine reduced eosinophil chemotaxis toward eotaxin when the eosinophils had been prestimulated with interleukin (IL)-5, but given alone it did not alter eosinophil chemotaxis toward IL-5. CD11b expression was inhibited when peripheral blood was prestimulated with IL-5, but eosinophil adhesion molecule expression was not altered. CONCLUSIONS: Epinastine suppresses allergic inflammation not only through its strong antihistamine and antimediator effects, but also by inhibiting eosinophilic chemotaxis and the expression of adhesion molecules involved in chemotaxis, especially CD11b.

Evidence for two components of object-based selection.

A wealth of research has shown that observers can bias visual processing toward specific locations, but the role of object-based selection is less clear In support of object-based selection, previous research has shown that when two objects are presented simultaneously, observers are better at reporting two attributes from one of the objects than one attribute from each object. However there has been controversy over whether this effect is best explained by object-based selection or spatial selection. Our work suggests that there are two separate components of selection in this task: (Ca) a spatial component that is observed when the relevant targets are cued for observers before the onset of the stimulus display and (b) an object-based component that can still be observed when the first component has been eliminated. The latter effect replicates the initial evidence in favor of object-based selection, and can be demonstrated even when the relevant targets are cued after the offset of the target stimuli.

Genetic diversity and biochemical characteristics of Trichosporon asahii isolated from clinical specimens, houses of patients with summer-type-hypersensitivity pneumonitis, and environmental materials.

Trichosporon asahii, which is distributed in the environment, is the major causative agent of the opportunistic infection trichosporonosis, and it also causes summer-type hypersensitivity pneumonitis (SHP). Random amplification of polymorphic DNA analysis was used to determine the intraspecies diversity of 39 T. asahii isolates from clinical specimens, SHP patients' houses, and environmental materials. The three primers used revealed 46 polymorphic bands. A phenogram was generated by the unweighted pair-group method with arithmetic mean. Clinical isolates formed a cluster, characterized by a 90% matching coefficient, but they did not cluster with strains isolated from SHP patients' houses or environmental sources. In addition, the biochemical characteristics of 86 strains from three sources were examined with 31 compounds using an ID32C kit, and a phenogram was constructed. The phenogram consisted of three major clusters. Cluster I included most of the clinical SHP isolates, and cluster II included most of the environmental isolates. Cluster III contained only one strain. A remarkable difference was found in the abilities of the strains belonging to clusters I and II to utilize six compounds. These results suggest that the genetic diversity and biochemical characteristics of T. asahii seem to be related to the source of the isolate. We also found a specific DNA fragment for the clinical isolates and strains isolated from SHP patients' houses.

Selective killing of human immunodeficiency virus-infected cells by targeted gene transfer and inducible gene expression using a recombinant human immunodeficiency virus vector.

A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector pseudotyped with HIV envelope containing the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the HIV LTR promoter (pHXTKN) was constructed and stably transferred into human CD4(+) H9, CEM, and U937 cells. RNase protection assays did not initially detect expression of the HSV-TK gene in HXTKN-transduced CD4(+) cells (HXTKN/CD4), but expression was then efficiently induced by infection with HIV-1. MTT assays showed that after HIV-1 infection, the susceptibility of HXTKN/CD4 cells to ganciclovir (GCV) was 1000-fold higher than prior to infection. This enabled HIV-1-infected cells to be selectively killed by transduction with HXTKN followed by exposure to GCV. Because the HSV-TK gene is specifically transferred into HIV-1-permissive cells and expressed only after HIV-1 infection, the frequency of unwanted cell death should be low. Elimination of the HIV-1-infected cells effectively inhibited further spread of infectious virus. In addition, the integrated HIV vector sequences were repackaged on infection with HIV-1 and transferred to surrounding untransduced cells. These results are indicative of the potential benefits of using HIV vectors in gene therapies for the treatment of HIV-1 infection.

2-Alkynyl-8-aryl-9-methyladenines as novel adenosine receptor antagonists: their synthesis and structure-activity relationships toward hepatic glucose production induced via agonism of the A(2B) receptor.

Novel adenosine antagonists, 2-alkynyl-8-aryl-9-methyladenine derivatives, were synthesized as candidate hypoglycemic agents. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. In general, aromatic moieties at the 8-position and alkynyl groups at the 2-position had significantly increased activity compared to unsubstituted compounds. The preferred substituents at the 8-position of adenine were the 2-furyl and 3-fluorophenyl groups. In modifying the alkynyl side chain, change of the ring size, cleavage of the ring, and removal of the hydroxyl group were well tolerated. The order of the stimulatory effects of adenosine agonists on rat hepatocytes was NECA > CPA > CGS21680, which is consistent with involvement of the A(2B) receptor. In Chinese hamster ovary cells stably transfected with human A(2B) receptor cDNA, one of the compounds potent in hepatocytes, 15o (IC(50) = 0.42 microM), antagonized NECA-induced stimulation of cyclic AMP production (IC(50) = 0.063 microM). This inhibitory effect was much more potent than those of FK453, KF17837, and L249313 which have been reported to be respectively A(1), A(2A), and A(3) selective antagonists. These findings agree very well with the result that, compared to 15o, these selective antagonists for each receptor subtype showed only marginal effects in rat hepatocytes. These results suggest that adenosine agonist-induced glucose production in rat hepatocytes is mediated through the A(2B) receptor. Furthermore, 15o showed hypoglycemic activity in an animal model of noninsulin-dependent diabetes mellitus, the KK-A(y) mice. It is possible that inhibition of hepatic glucose production via the A(2B) receptor could be at least one of the mechanisms by which 15o exerts its in vivo effects. Further elaboration of this group of compounds may afford novel antidiabetic agents.

Investigation of antisense DNA having C-5 polyamine substituted 2'-deoxyuridine derivative.

Phosphorothioate analogs of oligodeoxyribonucleotide (S-ODN) bearing non-branched polyamine molecule at C-5 position of certain pyrimidine base were synthesized. The synthesis of the modified S-ODNs was accomplished via post-synthetic modification method utilizing C-5 methoxycarbonylmethyl substituted deoxyuridine derivative with modest yields. The thermal stability of the dulexes containing modified S-ODNs was assessed through the measurement of the melting points (Tms). Interestingly, the Tms of the modified oligomers were considerably lower than that of the corresponding unmodified oligomer at relatively high concentration range. At lower concentration, on the other hand, the Tms of the modified S-ODNs were higher than that of the unmodified oligomer.

Targeted and stable gene delivery into muscle cells by a two-step transfer system.

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.

4-(3-Chloro-4-methoxybenzyl)aminophthalazines: synthesis and inhibitory activity toward phosphodiesterase 5.

We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1- and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) and their vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The preferred substituents at the 1-position of the phthalazine were 4-hydroxypiperidino, 4-hydroxymethylpiperidino, 4-(2-hydroxyethyl)piperidino, and 4-oxopiperidino. Among these compounds, [4-(3-chloro-4-methoxybenzyl)amino-1-(4-hydroxy)piperidino]-6-phthala zinecarbonitrile monohydrochloride (13) exhibited potent PDE5 inhibitory activity (IC(50) = 0.56 nM) with >1700-fold high selectivity over other PDE isozymes (PDE1-4). Compound 13 exhibited the most potent vasorelaxant action (EC(50) = 13 nM) in this series of compounds. Compound 13 reduced mean pulmonary arterial pressure by 29.9 +/- 3.1% when administered intravenously at 30 microg/kg to the chronically hypoxic rats and had an apparent oral bioavailability of about 19.5% in rats and was selected for further biological evaluation.

Overexpression of CD11b on eosinophils in atopic dermatitis: downregulation by cyclosporin A and upregulation by interleukin 5.

We examined the level of expression of CD11b on eosinophils in pripheral blood samples from patients with atopic dermatitis (AD) and non-AD volunteers. Eosinophils were defined using a new method employing CD14/CD45 and a backgate technique. Overexpression of CD11b was noted in eosinophils of AD patients. Treatment of AD with cyclosporin A resulted in clinical improvement as well as reduction in the expression of CD11b. Stimulation of eosinophils from patients with inactive AD by interleukin 5 upregulated the expression of CD11b on these cells. Our results suggest that the expression of CD11b surface molecule on eosinophils may play an important role in the activity of AD.

Muscle fiber immaturity and inactivity reduce myonecrosis in Duchenne muscular dystrophy.

We report on the first case of X-linked recessive myotubular myopathy (MTM1) coinciding with Duchenne muscular dystrophy (DMD). The muscle biopsy specimens of the patient show only the characteristic findings of MTM1, without the findings of DMD. We theorize that this was caused by the muscle fiber immaturity and inactivity.

[A sibling of delayed post-anoxic encephalopathy after strangulation].

A sibling of three year old girl and a year old boy, showed delayed post-anoxic encephlopathy after strangulation. After three days of the accident, the girl developed tetraplegia and choreo-athetosis. Her brother also developed choreo-athetosis two weeks after strangulation. T2 weighted MRI revealed a high signal intensity in the bilateral putamen and caudate nucleus. After hyperbaric oxygen therapy for two months, their symptoms diminished. We hypothesize that the functional damage of the neurons occurred in the bilateral basal ganglia as delayed neuronal death because of their vulnerability and peculiarity of the local circulation. Hyperbaric oxygen therapy may be effective in rescuing the neurons from hypoxia.

4-Benzylamino-1-chloro-6-substituted phthalazines: synthesis and inhibitory activity toward phosphodiesterase 5.

We synthesized various 4-benzylamino-1-chloro-6-substituted phthalazines (15) and 4-benzylamino-1-chloro-7-substituted phthalazines (16) and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) purified from porcine platelets. The PDE5-inhibitory activities of 15 were greater than those of the isomers (16). The preferred substituent at the 4-position of phthalazine was a (3-chloro-4-methoxybenzyl)amino group, and those at the 6-position were cyano, nitro, and trifluoromethyl groups. Compounds 15a (IC50 = 4.8 nM), 15f (3.5 nM), and 15i (5.3 nM) were more potent inhibitors than E4021 (8.6 nM). Compounds 15a and 15f also showed vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The EC50 values for vasorelaxant action of 15a, 15f, and E4021 were 150, 160, and 980 nM, respectively. These results show that novel PDE5 inhibitors possessing a potent vasorelaxant effect may exist among phthalazine derivatives.

Enhanced production of RANTES, an eosinophil chemoattractant factor, by cytokine-stimulated epidermal keratinocytes.

In allergic skin diseases such as atopic dermatitis (AD), eosinophils migrate from the circulation to the skin. We investigated the mechanisms of eosinophil chemotaxis in atopic dermatitis by examining the effect of stimulation of epidermal keratinocytes (KC) by inflammatory cytokines, interferon-gamma (IFNgamma) and/or tumor necrosis factor-alpha (TNF alpha) on the production of eosinophil chemotactic factors. Simultaneous addition of IFNgamma and TNF alpha to culture KC synergistically increased eosinophil chemotaxis and the expression of RANTES mRNA and protein level on these cells. Anti-RANTES antibody blocked eosinophil chemotaxis by IFNgamma- and TNF alpha-stimulated KC. Our results indicate that the production of RANTES by KC may help to explain eosinophil infiltration into the skin in AD.

Steroid-induced changes of eosinophils in atopic dermatitis.

We investigated whether apoptosis of eosinophils is specific to atopic dermatitis (AD), or also occurs in other diseases with eosinophilia. We examined the survival of eosinophils cultured with corticosteroids: (1) Clinically, steroid administration significantly decreased high peripheral blood eosinophil cell counts in patients with AD. (2) Treatment with recombinant human (rh) IL-5 prolonged the life span of eosinophils derived from patients with AD and of those derived from non-AD patients with eosinophilia. However, there were differences in the survival rates in the presence of rhIL-5: the eosinophils from non-AD patients showed 1.4-fold higher survival rates than those from AD patients at 24 h. In the presence of steroids, the eosinophils from non-AD patients showed a survival rate double that of those from AD patients at 24 h. (3) In eosinophils from patients with AD, the survival rate decreased significantly in a time- and steroid-concentration-dependent manner. Steroid administration significantly inhibited the survival rate of eosinophils from patients with AD compared to those of monocytes and neutrophils. These findings suggest that apoptosis induced by steroids decreases the eosinophil count in vivo in patients with AD. There may be a difference in the incidence of steroid-induced apoptosis between eosinophil cells from patients with AD and those from patients with eosinophilia due to other underlying diseases.

Synthesis and anti-HIV activity of phosphorothioate antisense DNA containing C-5 polyamine substituted 2'-deoxyuridine and/or acridine residues.

Novel phosphorotioate DNAs (S-ODNs) bearing polyamine moiety at the C-5 position of 2'-deoxyuridine in place of certain thymidine residues and/or an acridine moiety at the 5'-terminus were prepared. The sequence of the S-ODNs is complementary to the rev region of HIV-1 mRNA. The duplexes consisted of the modified S-ODNs and the complementary DNA exhibited the enhanced stability compared to that consisted of the corresponding unmodified S-ODN and the complement. The improved antisense activity against HIV-1 was also observed for all modified S-ODNs.

Immunohistology of skin and oral biopsies in graft-versus-host disease after bone marrow transplantation and cytokine therapy.

BACKGROUND: Early diagnosis of graft-versus-host-disease (GVHD) after bone marrow transplantation is often difficult, particularly when the patients are immunosuppressed by chemotherapy or irradiation. OBJECTIVE: To investigate the influence of cytokines on skin lesions after bone marrow transplantation. METHODS: Biopsy specimens of skin and oral mucosa were obtained from bone marrow transplant patients with GVHD and were subjected to histologic and immunohistochemical examination. RESULTS: Administration of granulocyte-macrophage colony-stimulating factor caused atopic dermatitis-like lesions in two patients, who had infiltration of neutrophils, eosinophils, and lymphocytes around the hair follicles of the skin and no signs of GVHD in other organs. Only patients who were treated with cytokines developed acute GVHD. Immunohistochemical examination of skin biopsies from 18 patients with acute GVHD and 11 patients with chronic GVHD after cyclophosphamide administration or irradiation showed that the maculopapular skin lesions characteristic of acute GVHD contained infiltrates of CD4+ and CD8+ lymphocytes. There was also an increase in numbers of epidermal keratinocytes expressing intercellular adhesion molecule-I and HLA-DR antigens. CONCLUSION: These findings support the involvement of cytokines in GVHD and suggest that immunostaining of skin biopsies may be useful for the early diagnosis of this condition.

RANTES mRNA expression in skin and colon of patients with atopic dermatitis.

The expression of RANTES mRNA in dermal and colonic tissue was examined in patients with atopic dermatitis by the reverse transcription polymerase chain reaction method. RANTES mRNA was detected in the colon in 8 of 10 patients and in 1 of 5 control patients. It was present in rashes in 9 of 10 patients and at non-eruptive sites in 5 of 7 patients. These findings suggest that RANTES is involved in eosinophil infiltration and T cell infiltration in atopic dermatitis.

Corticosteroid-induced apoptosis of eosinophils in atopic dermatitis patients.

We examined the mechanism by which steroid administration significantly decreases the high eosinophil cell count in peripheral blood in patients with atopic dermatitis (AD). Eosinophils were isolated from the peripheral blood of patients with moderate or severe adult AD, and cultured. After steroid was added to the culture medium, we examined the changes in eosinophils, i.e., 1) the survival rates, 2) morphological changes and 3) fragmentation of DNA with respect to 2 factors, the steroid concentration and culture time. The steroid or interleukin-5 (IL-5) were added to eosinophils from patients with AD and those from non-AD patients with eosinophilia to compare serial changes in the survival rate. In eosinophils from patients with AD, the survival rate significantly decreased time-dependently and steroid concentration-dependently. Steroid administration significantly inhibited the survival rate of eosinophils from patients with AD compared to the survival rates of monocytes and neutrophils. The nuclei of eosinophils were serially reduced, and disappeared 72 hours after steroid administration. Simultaneously, cell size decreased, although the cell membrane remained intact. Granules developed in the cell membrane. In the steroid-treated group, apoptotic cells appeared earlier than in the untreated group. The number of cells showing apoptosis was increased steroid concentration-dependently. The number of DNA ladders was increased time-dependently and steroid concentration-dependently. In eosinophils derived from patients with AD and those derived from non-AD patients with eosinophilia, treatment with recombinant human (rh) IL-5 prolonged the life-span of cells. However, there were differences in the survival rates. In the presence of rhIL-5, the eosinophils from non-AD patients survived 1.4 times longer than those from AD patients at 24 hours (P < 0.05). In the presence of steroid, the eosinophils from non-AD patients survived twice as long as those from AD patients at 24 hours (P < 0.01). These findings suggest that apoptosis induced by steroids decreases the eosinophil count in vivo in patients with AD. There may be a difference in the incidence of steroid-induced apoptosis between eosinophil cells from patients with AD and those from patients with eosinophilia due to other underlying diseases.

Elevated plasma RANTES levels in patients with atopic dermatitis.

Eosinophils and T cells are involved in the pathologic process of atopic dermatitis. To further understand the role of these cells, and the possible involvement of RANTES, in the pathogenesis of atopic dermatitis, we measured the plasma level of RANTES using the sandwich ELISA. The mean plasma level of RANTES in 11 patients with atopic dermatitis was significantly higher than that of 15 normal control subjects. RANTES levels were higher in patients with severe form of atopic dermatitis than that of patients with mild disease. These findings suggest that RANTES may play a role in the recruitment and activation of eosinophils and T cells in atopic dermatitis.

In vivo inhibition of hepatitis B virus gene expression by antisense phosphorothioate oligonucleotides.

While an important goal of treatment for hepatitis B is to prevent the development of hepatocellular carcinoma, there has been no effective therapy for it. Antisense oligodeoxynucleotide treatment could in principle inhibit hepatitis B virus gene expression and suppress tumor development. We used a mouse model for hepatocellular carcinoma, which is transgenic for the hepatitis B virus HBx gene, to study antisense phosphorothioate oligodeoxynucleotides. Among 2 series of sense and antisense oligodeoxynucleotides, only antisense sequences covering the initiation codon of the HBx gene effectively inhibited the expression of the HBx gene in the liver. Intraperitoneal injection of this antisense oligodeoxynucleotide thrice a week for 8 weeks resulted in the prevention of preneoplastic lesion development in the liver without inflammation in the liver or developmental disturbance of the mice. Antisense phosphorothioate oligodeoxynucleotides can inhibit the expression of a hepatitis B virus gene and may be a promising method for the prevention of hepatocellular carcinoma in hepatitis B virus infection.