BACKGROUND: Posttransfusion graft-versus-host disease (PT-GVHD) is a pathophysiological process of the immune system. Mature donor T cells that have survived the host defense mechanisms recognize recipient allo-antigens through antigen-specific T-cell receptors and mount an immune attack against certain host antigens. STUDY DESIGN AND METHODS: To characterize the T cells involved in PT-GVHD, expression of the T-cell receptor V beta genes in peripheral blood T cells isolated from four PT-GVHD patients was analyzed. T-cell receptor beta chain cDNA derived from mRNA extracted from peripheral blood lymphocytes was amplified by an inverse polymerase chain reaction method, and the cDNA was subsequently cloned and sequenced. RESULTS: The cDNA clones derived from patients with PT-GVHD showed remarkable differences in the distribution of T-cell receptor V beta gene use from that in normal controls. Very restricted T-cell receptor V beta gene repertoires and preferential expression of certain V beta gene segments were revealed in the peripheral blood lymphocytes of each patients. Shared V beta gene use among the patients was not observed, probably because of HLA differences among the patient-donor combinations. CONCLUSION: This restricted use reflected in vivo expansion of a limited number of engrafted T-cell clones at the onset of PT-GVHD and suggested that those oligoclonal donor T cells may be involved in the induction of PT-GVHD.

Aged↗

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Inhibition of influenza virus replication by phosphorothioate and liposomally endocapsulated oligonucleotides.

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. Phosphorothioate and liposomally encapsulated oligonucleotides with two target sites (PB1 and PB2) were synthesized and tested for virus-induced cytopathogenicity effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODNs complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODNs targeted to PB1 was considerably decreased in comparison with the PB2 target sites. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the modified oligonucleotides are effectively enhanced by using the liposomal carrier.

Antiviral Agents↗

PubMedDate not supplied

In vitro and in vivo anti-influenza A virus activity of antisense oligonucleotides.

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.

Animals↗

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Antisense oligodeoxynucleotide complementary to CXCR4 mRNA block replication of HIV-1 in COS cells.

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatment of acquired immunodeficiency syndrome, we examined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation initiation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2 microM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natural phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This study defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.

Animals↗

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Antisense phosphorothioate oligonucleotides targeted to the human chemokine receptor CXCR4.

The CXC chemokine receptor CXCR4 is used as a major co-receptor for fusion and entry by syncytia-inducing T-tropic (X4) isolates of HIV-1. In the present study, we report the effects of an antisense oligodeoxyribonucleotide on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for Human Immunodeficiency Virus type 1 (HIV-1) infection. Antisense phosphorothioate oligodeoxyribonucleotides (anti-S-ODNs) corresponding to the sequence of bases 69 to 88 of the human CXCR4 mRNA gene were synthesized. When the naked anti-S-ODN was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor were reduced up to 50%, indicating sequence-specific inhibition. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for delivery of anti-S-ODNs. The anti-S-ODN encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN.

Base Sequence↗

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Anti-HIV-1 activity by a triple-helix forming oligonucleotides targeted to polypurine tract on viral RNA.

Reverse transcription of HIV-1 into double-stranded DNA involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by reverse transcriptase (RT). The PPT is a possible target for triple-helix formation. We show the effects of triple-helix formation by assays of RNase H cleavage inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RNase H activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RNase H. The FTFOs containing the phosphorothioate groups at the antisense strand showed greater 3'-exonuclease resistance. In HIV-1 infected MT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phosphorothioate oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Anti-HIV Agents↗

PubMedDate not supplied

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleaveage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

Animals↗

PubMedDate not supplied

22-Oxacalcitriol upregulates p21(WAF1/Cip1) in human parathyroid glands. A preliminary report.

In the era of 22-oxacalcitriol (OCT), newly synthesized 1 alpha,25-dihydroxyvitamin D(3) analogue, against secondary hyperparathyroidism, the indications of parathyroidectomy (PTx) has been restricted. Recent investigations on animal models have revealed the inhibitory effects on PTH secretion after OCT treatment, whereas there has been no evidence about human parathyroid glands. A 38-year-old man with a 19-year history of hemodialysis was performed PTx after the failure of OCT treatment. Expressions of proliferative nuclear cell antigen (PCNA), calcium-sensing receptor (CaSR), vitamin D receptor (VDR), p53 and p21(WAF1/Cip1) were analyzed by Western blotting and immunohistochemistry on resected parathyroid glands. We confirmed up-regulations of CaSR and VDR, which contribute the reduction of serum PTH, by OCT treatment. Concomitant up-regulation of p21(WAF1/Cip1) but not p53, especially in nodular hyperplasia, can be considered to induce cell cycle arrest of the parathyroid cells, but not cytocidal effect of OCT.

Adult↗

PubMedDate not supplied

Pharmacokinetic analysis of the disposition of valproate in pregnant rats.

Kinetic analysis of valproate (VPA) disposition in pregnant rats was performed. A dose-dependent saturable plasma elimination was observed in both the control and pregnant rats. Saturation was more remarkable in the pregnant rats and this was assumed to be due to the decreased hepatic extraction by metabolism. Pharmacokinetic parameters were calculated using a two-compartment open model with a Michaelis-Menten-type elimination process from the central compartment. In the pregnant rats, the calculated values of Km and Vmax decreased significantly, by one-half of those of the control rats, whereas no significant difference was observed in distribution rate constants (k12, k21) and volumes of distribution (V1, Vdss) between two groups of rats. Furthermore, the hepatic extraction of VPA in the control and pregnant rats was investigated in order to explain nonlinear pharmacokinetics of VPA. The values of the hepatic extraction ratio in the control and pregnant rats were 39.9 and 22.4% at the steady state concentration of approximately 20 micrograms/ml, respectively. The extraction ratios in the pregnant rats were lower than those of the control rats. This fact may be one of the reasons why the elimination of VPA in the pregnant rats is more susceptible to saturation.

Animals↗