Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A.

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.

Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Ceramide limits phosphatidylinositol-3-kinase C2ß-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

Thiol proteinase inhibitors reverse the increased protein kinase C down-regulation and concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mouse.

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2(+)-dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.

Low-density lipoprotein oxidized by polymorphonuclear leukocytes inhibits natural killer cell activity.

We studied the effect of low-density lipoprotein (LDL) oxidized by opsonized zymosan-stimulated polymorphonuclear leukocytes (PMN) on natural killer (NK) cell activity. Oxidized LDL inhibited NK cell activity in a dose-dependent manner, whereas normal LDL left it unaffected. However, oxidized LDL did not inhibit antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, a positive correlation was observed between the amount of thiobarbituric acid-reacting substances (TBARS) on the sample of oxidized LDL and the degree of inhibition of NK cell activity. We also showed that oxidized LDL suppressed the binding capacity of purified large granular lymphocytes (LGL) to target cells without changing the lytic activity. These results therefore suggest that activated PMN can modulate NK cell activity by oxidizing LDL.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation.

BACKGROUND: In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. METHODS: Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. RESULTS: Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. CONCLUSIONS: In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Cloning and characterization of a new allergen, Mag 3, from the house dust mite, Dermatophagoides farinae: cross-reactivity with high-molecular-weight allergen.

A new immunoreactive clone whose sequence is not homologous to that of any of the previously identified mite allergens was isolated by successive immunoscreening of a Dermatophagoides farinae cDNA library with rabbit antisera to an extract of the house dust mite and IgE in pooled sera from patients allergic to mites. Rabbit antibodies specific for the recombinant protein recognized a 177 kD protein in a mite body extract. This immunoreactive protein was located in the circumferential tissues of esophagus, gut and the other internal organs in mites. The reaction of human IgE to the purified natural antigen was inhibited competitively to 30% by the recombinant antigen. In terms of the frequency and the intensity of response to specific IgE in sera from asthmatic patients, the natural protein was similar to Der f2, while the recombinant protein was slightly less allergenic by these criteria. We conclude that the natural protein from the house dust mite, D. farinae, is an important allergen.

Anti-angiogenesis agent DS-4152 is a potent and selective inhibitor of HIV-1 replication in vitro.

OBJECTIVE: To determine whether the anti-angiogenesis agent DS-4152 inhibits the replication of HIV-1 in vitro. DESIGN: A sulfated polysaccharide-peptidoglycan DS-4152 has recently been identified as a potent and selective inhibitor of Kaposi's sarcoma (KS). Therefore, it is important to evaluate the anti-HIV-1 activity of DS-4152 alone and in combination with dideoxynucleosides. METHODS: Activity of DS-4152 against HIV-1 replication was examined in MT-4, Molt-4, and peripheral blood lymphocyte cells. The inhibitory effect of the compound on syncytium-formation was determined by cocultivation of Molt-4 cells with Molt-4/IIIB cells. Inhibition of virus adsorption to the host cells was measured by a p24 antigen capture enzyme-linked immunosorbent assay. RESULTS: DS-4152 showed potent and selective inhibition of HIV-1 replication in the cell systems. Its 50% effective concentration for HIV-1 (IIIB strain) in MT-4 cells was 0.7 microgram/ml. The compound was not cytotoxic at concentrations < or = 100 micrograms/ml. DS-4125 proved inhibitory to syncytium-formation and virus adsorption. The anti-HIV-1 activities of zidovudine, dideoxycytidine and dideoxyinosine were not affected by the presence of DS-4152. CONCLUSION: DS-4152 has the potential, from these in vitro studies, to function as an anti-HIV-1 as well as an anti-angiogenesis agent. In order to determine this possibility, consequences of DS-4152 infusion on HIV-1 p24 serum levels and CD4+ cell counts over time are being examined in ongoing clinical trials in the United States on patients with AIDS-associated KS.

Follicular dendritic cells in vitro are not susceptible to infection by HIV-1.

OBJECTIVES: To investigate whether follicular dendritic cells (FDC) are target cells for HIV-1 infection. DESIGN: Based on the principle that if FDC are true target cells, HIV-1 particles will bind to the surface of FDC and then invade their cytoplasm and nuclei. METHODS: Freshly isolated tonsilar FDC were exposed to two strains (HE and JR-FL) of HIV-1 and cultured. They were then examined for HIV-1 replication, using p24 antigen-capture enzyme-linked immunosorbent assay, immunolabelling, and polymerase chain reaction (PCR) amplification. We used FDC clusters as the FDC source, since the culture system for FDC clusters has various advantages over other methods for the successful long-term culture of FDC. RESULTS: After 2 h incubation, particles of both HIV-1 strains bound to the surfaces of FDC, as well as to CD4+ T cells, although FDC do not have CD4 receptors. The FDC gradually released the particles into the culture supernatant. More HIV-1 particles were bound to fresh FDC than to dedifferentiated FDC or to control fibroblasts. However, HIV-1 particles bound to the FDC did not seem to enter the cells. We found no evidence of HIV-1 proviral DNA synthesis in FDC. CONCLUSIONS: Our results suggest that FDC are not readily infected with HIV-1 in situ, although we found that FDC in vitro were not infected by HIV-1.

Selective inhibition of human immunodeficiency virus type 1 replication by novel fluoroalkylated oligomers in vitro.

Several fluoroalkylated oligomers were found to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) in vitro. Among the test compounds, bis(perfluoro-1,4,7,10-tetramethyl-2,5,8,11-tetraoxatetradecyl+ ++)methacrylic acid oligomer (MAA-HFPO5) emerged as the most potent inhibitor of HIV-1 replication. Its 50% antivirally effective concentration for the IIIB strain was 2.8 mu g/ml, whereas the compound did not affect the growth and viability of mock-infected MT-4 cells at concentrations < or = 100 micrograms/ml. MAA-HFPO5 was also inhibitory to other strains of HIV-1 in various human T-cell systems, including peripheral blood lymphocytes. MAA-HFPO5 inhibited syncytium formation and virus adsorption. The combination of MAA-HFPO5 with either 3'-azido-3'dioxythymidine or dextran sulfate resulted in an additive effect. Thus, fluoroalkylated oligomers are novel HIV-1 inhibitors that warrant further evaluation of their therapeutic potential.

Nucleosides and nucleotides. 185. Synthesis and biological activities of 4'alpha-C-branched-chain sugar pyrimidine nucleosides.

A series of 4'alpha-C-branched-chain pyrimidine nucleosides was synthesized from 2'-deoxycytidine or uridine. In the 2'-deoxycytidine series, the substituent at the 4'alpha-position affected cytotoxicity against L1210 mouse leukemic cells in vitro in the order Me (23) > CN (22) > C(symbol)CH (21) > CH=CH(2) (19) > Et (24) > CH=CHCl (20). However, uridine and cytidine derivatives with ethynyl and cyano groups at the 4'alpha-position did not show any cytotoxicity. The antiviral activities of these nucleosides against HSV-1, HSV-2, and HIV-1 in vitro were also examined. Compounds 22 and 23 showed antiviral activities against HSV-1 and HSV-2 without showing significant toxicity to the host cells (MRC-5 cells). Although almost all of the nucleosides showed anti-HIV-1 activities, they were also cytotoxic to the host cells (MT-4).

A new class of HIV-1-specific 6-substituted acyclouridine derivatives: synthesis and anti-HIV-1 activity of 5- or 6-substituted analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT).

A series of novel acyclouridine derivatives substituted at both the C-5 and C-6 positions were synthesized for the purpose of improving the activity of a recently reported HIV-1-specific lead, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). Preparation of C-6 substituted derivatives was carried out based on the following three methods: (1) LDA (lithium diisopropylamide) lithiation of a thymine derivative (4) and subsequent reaction with electrophiles, (2) an addition-elimination reaction of HEPT or its 6-(phenylsulfinyl) derivative (10), or (3) palladium-catalyzed cross-coupling between a 6-iodo derivative (16) and terminal alkynes. Following the methods, 21 C-6 substituted analogues were synthesized. Among these, 6-(cyclohexylthio) (8), 6-phenoxy (13), and 6-benzyl (27) derivatives showed anti-HIV-1 (HTLV-IIIB) activity with EC50 values of 8.2, 85, and 23 microM, respectively. Preparation of C-5 substituted derivatives was based on either LTMP (lithium 2,2,6,6-tetramethylpiperidide) lithiation of 6-(phenylthio)uracil derivative 37 or the above mentioned palladium-catalyzed cross-coupling of a 5-iodo-6-(phenylthio)uracil derivative (38). Following these methods, 11 C-5 substituted analogues were synthesized. Some 5-substituted derivatives (5-I, 44; 5-CH = CPh2, 49; 5-CH = CHPh (Z), 54; and 5-CH = CH2, 55) were more active than HEPT, but their selectivity indices (SI = CC50/EC50) were lower than that of HEPT. Compound 8 was also evaluated against another HIV-1 strain (HTLV-IIIRF) and HIV-2 strains (LAV-2ROD and LAV-2EHO). Only HTLV-IIIRF was as sensitive as HTLV-IIIB.

Synthesis and anti-HIV activity of 2-, 3-, and 4-substituted analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT).

Several analogues of a new lead for anti-HIV-1 agents, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), in which the C-2, N-3, or C-4 position was modified were synthesized. These involve 2-thiothymine (11), 2-thiouracil (12), 4-thiothymine (17), 4-thiouracil (18), 5-methylcytosine (27), and cytosine (28) derivatives. Preparation of N-3-substituted derivatives (29 and 30) of HEPT was also carried out. Among these analogues, compound 11 exhibited excellent activity against HIV-1 HTLV-IIIB strain with an EC50 value of 0.98 microM, which is 7-fold more potent than that of HEPT. Removal of the 5-methyl group in compound 11 results in total loss of activity. Other compounds did not show any anti-HIV-1 activity. The 4-thio derivatives 17 and 18 were found to be rather cytotoxic. When compound 11 was evaluated for its inhibitory effects on another HIV-1 strain, HTLV-IIIRE, and two HIV-2 strains, LAV-2ROD and LAV-2EHO, it proved equally inhibitory to HTLV-IIIRF, whereas both HIV-2 strains were insensitive to the compound.

Synthesis and antiviral activity of deoxy analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents.

The effect of substitution in the acyclic structure of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) on anti-HIV-1 activity was investigated by synthesizing a series of deoxy analogs and related compounds. Preparation of 1-[(2-alkyloxyethoxy)methyl]-6- (phenylthio)thymine (2-4) derivatives was carried out based on alkylation of HEPT with primary alkyl halides. Preparation of the 1-[(alkyloxy)methyl]-6-(phenylthio)thymine (26-31) and 1-[(alkyloxy)methyl]-6-(arylthio)-2-thiouracil (32-45) derivatives was carried out on the basis of LDA lithiation of 1-[(alkyloxy)-methyl]thymine (9-14) and 1-[(alkyloxy)methyl]-2-thiouracil (15-25) followed by reaction with diaryl disulfides. The oxidative hydrolysis of the 2-thiouracil derivatives gave 1-[(alkyloxy)methyl]-6-(arylthio)uracil derivatives (46-57). 1-Alkyl-6-(phenylthio)thymine (59-61) derivatives were prepared on the basis of alkylation of 6-(phenylthio)thymine (58). Methylation of the hydroxyl group of HEPT did not affect the anti-HIV-1 activity of HEPT. Substitution of the 1-(2-hydroxyethoxy)methyl group by ethyl, butyl, methoxymethyl, (propyloxy)methyl, and (butyloxy)-methyl groups somewhat improved the original anti-HIV-1 activity of HEPT. Substitution with ethoxymethyl and (benzyloxy)methyl groups further potentiated the activity [EC50: 1-(ethoxy-methyl)-6-(phenylthio)thymine (27), 0.33 microM; 1-[(benzyloxy)methyl]-6-(phenylthio)thymine (31), 0.088 microM]. When the 5-methyl group of 27 and 31 was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity was improved remarkably [EC50: 5-ethyl-1-(ethoxymethyl)-6-(phenylthio)-uracil (46), 0.019 microM; 5-ethyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (52), 0.0059 microM; 5-isopropyl-1-(ethoxymethyl)-6-(phenylthio)uracil (55), 0.012 microM; 5-isopropyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (56), 0.0027 microM]. Introduction of two m-methyl groups into the phenylthio ring also potentiated the activity.

Synthesis and antiviral activity of 6-benzyl analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents.

Several 6-benzyl analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (1; HEPT) were synthesized and evaluated for their anti-HIV-1 activity. LDA (lithium diisopropylamide) lithiation of 5-ethyluracil derivatives 7 and 8 and subsequent reaction with an aryl aldehyde gave 6-(arylhydroxymethyl)-5-ethyluracil derivatives 9-12. 6-(Arylhydroxymethyl)-5-isopropyluracil derivatives 15-18 were prepared from the 5-isopropyl-2-thiouracil derivatives 13 and 14 by the above procedure following oxidative hydrolysis of the thione. Preparation of the target 5-alkyl-1-(alkoxymethyl)-6-benzyluracil derivatives 27-34 was carried out by acetylation of 9-14 followed by Pd-catalyzed hydrogenolysis. The 1-butyl- (37 and 39) and 1-(2-methoxyl)- (38 and 40) 5-alkyl-6-benzyluracils were synthesized by 1-alkylation of the 3-phenacyl derivatives 35 and 36 with alkyl halides followed by deprotection of the 3-phenacyl group. Compounds synthesized in this study inhibited HIV-1 replication in MT-4 cells in the submicromolar to namomolar concentration range. From this series of compounds, 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (33) was selected for clinical evaluation.

Structure-activity relationships of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine analogues: effect of substitutions at the C-6 phenyl ring and at the C-5 position on anti-HIV-1 activity.

The effect of substitution on the pyrimidine moiety of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine (HEPT-S) on anti-HIV-1 activity was investigated by synthesizing a series of 5-methyl-6-(arylthio) and 5-substituted-6-(phenylthio) derivatives. Preparation of the 5-methyl-6-(arylthio) derivatives was carried out based on either LDA lithiation of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]thymine (3) and 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiothymine (4) followed by reaction with diaryl disulfides or an addition-elimination reaction of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]-methyl]-6- (phenylsulfinyl)thymine (31) with aromatic thiols. Preparation of the 5-substituted-6-(phenylthio) derivatives was carried out based on either C-5 lithiation of the 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-6-(phenylthio)uraci l (41) with LTMP or the LDA lithiation of 5-alkyl-1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiouraci l derivatives 45-47. Substitution at the meta position of the C-6-(phenylthio) ring by the methyl group improved the original anti-HIV-1 activity of HEPT, and introduction of two m-methyl groups to the phenylthio ring further potentiated the activity [EC50: 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]thymine (28), 0.26 microM; 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]-2-thiothymin e (30), 0.22 microM]. When the 5-methyl group was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity of HEPT was also improved remarkably [EC50: 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (48), 0.11 microM; 5-isopropyl-1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)-2-thiouracil (50), 0.059 microM; 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (54), 0.12 microM; 5-isopropyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (56), 0.063 microM]. 6-[(3,5-Dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]thymine derivatives 51 and 57 and 6-[(3,5-dimethylphenyl)thio]-5-isopropyl-1-[(2- hydroxyethoxy)methyl]thymine derivatives 52 and 58 inhibited the replication of HIV-1 in the nanomolar concentration range.

Syntheses of 4'-C-ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosylpyrimidines and -purines and evaluation of their anti-HIV activity.

4'-C-Ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosylpyrimidine and -purine nucleosides were synthesized and evaluated for their in vitro anti-HIV activity. The key intermediate, 4-C-ethynyl- or 4-C-triethylsilylethynyl-D-ribo-pentofuranose, was prepared from D-glucose and glycosidated with various pyrimidine or purine bases. The arabinopyrimidine derivatives were prepared from the corresponding ribo derivatives via O(2),2'-anhydro nucleosides. The 2'-deoxy-ribo derivatives were synthesized by radical reduction of 2'-bromo or 2'- phenoxythiocarbonyloxy nucleosides. Among these 4'-C-ethynyl nucleosides, seven analogues proved to be potent against HIV-1 in vitro with EC(50) values ranging from 0.0003 to 0. 03 microM. These compounds also exerted activity against clinical and multi-dideoxy-nucleoside-resistant HIV-1 strains with comparable EC(50) values. Three such 4'-C-ethynyl-2'-deoxypurine analogues including 4'-C-ethynyl-2'-deoxyadenosine and 4'-C-ethynyl-2, 6-diamino-2'-deoxypurine were less cytotoxic [selectivity indices (SIs): 975-2733] than three 4'-C-ethynyl-2'-deoxycytidine analogues (SIs: 63-363). 4'-C-Ethynyl-5-fluoro-2'-deoxycytidine was least toxic (SI: >3333) and potent against all HIV strains tested.

B cell mitogenic activity of house dust mite, Dermatophagoides farinae, antigens.

The effect of mite antigens on murine and human lymphocytes was studied in vitro. Antigens prepared from Dermatophagoides farinae feces and bodies stimulated normal murine spleen cells to proliferate in a dose-dependent manner. The responder cells are B cells, because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement, but not with anti-Thy 1 antibody and complement. Furthermore, nylon column-purified T cells did not respond. The stimulation of B cells with mite antigens was not due to the contamination of lipopolysaccharide, a representative B cell mitogen, because C3H/HeJ spleen cells which are low responders to lipopolysaccharide could respond to mite antigens. These antigens induced not only proliferative response of murine B cells, but also immunoglobulin production. By gel-filtration column chromatography, the active fractions were eluted around the molecular weight of 150-155 kDa. Furthermore, mite antigens also stimulated human B cells to proliferate and to produce immunoglobulin. All these results suggest that mite antigens are a potent B cell mitogen and this activity might concern the induction of allergic reaction.

T-cell epitope analysis of Mag 3, an important allergen from the house dust mite, Dermatophagoides farinae.

Here we describe the detection of T-cell epitope region on the house dust mite allergen Mag 3, which has been shown to trigger T-cell proliferation in mite-allergic asthmatic patients. We first examined murine T-cell epitope using T-cell fraction prepared from recombinant Mag 3 (r-Mag 3)-primed H-2k mice. Initial proliferation assay with truncated r-Mag 3 indicated that N-terminal 113 amino acid region was required for triggering T-cell activation. Subsequent epitope scanning with synthetic overlapping peptides revealed that T-cell reactive region was assigned within amino acid range 56-75. We also explored human T-cell determinant using specific T-cells from mite-allergic patients. Intriguingly, we found that amino acid range 56-85, a portion partially overlapping with that identified in r-Mag 3-primed mice, was exclusively recognized by T-cells from different patients. Further investigation of unique T-cell epitope region found in this study would provide insight into the development of animal therapeutic model and/or peptide vaccine for asthma.