ABSTRACT
The objective of this work was to determine if fluorescence-activated cell sorting (FACS) can be used to isolate trophoblast giant cells (TGCs) from bovine placentae. Cotyledons were harvested, minced and digested with dispase/pancreatin. Tissue homogenates were incubated with Vybrant Dye Cycle-Green. FACS was completed, and cells with DNA content 2- to 4-times greater than cells within the diploid peak contained between 65 and 84% TGCs. The relative abundance of CSH1 and PAG1 transcripts were greater (P < 0.05) in TGC-enriched fractions than cells within the diploid peak. These observations indicate that FACS can effectively enrich TGCs from bovine placentae.
Subject(s)
Trophoblasts/cytology , Animals , Cattle , Female , Flow Cytometry , PregnancyABSTRACT
Peptide T, which is derived from the V2 region of HIV-1, inhibits replication of R5 and dual-tropic (R5/X4) HIV-1 strains in monocyte-derived macrophages (MDMs), microglia, and primary CD4(+)T cells. Little to no inhibition by peptide T was observed with lab adapted X4 viruses such as IIIB, MN, or NL4-3 propagated in CD4(+) T cells or in the MAGI entry assay. The more clinically relevant R5/X4 early passage patient isolates were inhibited via either the X4 or R5 chemokine receptors, although inhibition was greater with R5 compared to X4 receptors. Virus inhibition ranged from 60 to 99%, depending on the assay, receptor target, viral isolate and amount of added virus. Peak inhibitory effects were detected at concentrations from 10(-12) to 10(-9) M. Peptide T acted to block viral entry as it inhibited in the MAGI cell assay and blocked infection in the luciferase reporter assay using HIV virions pseudotyped with ADA envelope. These results using early passage virus grown in primary cells, together with two different entry reporter assays, show that peptide T selectively inhibits HIV replication using chemokine receptor CCR5 compared to CXC4, explaining past inconsistencies of in vitro antiviral effects.
Subject(s)
HIV-1/physiology , Peptide T/physiology , Receptors, CCR5/physiology , Virus Replication/drug effects , Antibodies, Monoclonal/immunology , Antiviral Agents/metabolism , Biological Assay , Cells, Cultured , Chemokines/antagonists & inhibitors , Chemokines/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Fetus , Flow Cytometry , Genes, Reporter , HIV Core Protein p24/immunology , HIV-1/metabolism , HeLa Cells , Humans , Luciferases/analysis , Luciferases/genetics , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Microglia/cytology , Microglia/metabolism , Microglia/virology , Peptide T/immunology , Time FactorsABSTRACT
OBJECTIVE: To determine the influences on pediatric AIDS of a heterozygous 32 base pair deletion in the CC-chemokine receptor 5 gene (CCR5 wt/Delta 32) and a common polymorphism in the 3' untranslated region of stromal cell-derived factor-1 beta gene transcript (SDF1-3'A). DESIGN: The rate of HIV-1 disease progression and viral burden were compared according to the CCR5 and SDF-1 genotypes in 127 (58 Caucasians, 60 African-Americans and nine Hispanics) perinatally HIV-1-infected children. RESULTS: Regardless of ethnic background, the CCR5 wt/Delta 32 genotype was associated with a delayed onset of AIDS-defining infectious complications during the first 5 years of infection [relative hazard (RH) = 0.22; 95% confidence interval (CI), 0.012--1.02; P = 0.053]. Similarly, CCR5 wt/Delta 32 conferred an early protection against severe immune suppression and HIV-1 encephalopathy, but only in those without SDF1-3'A (RH = 0; 95% CI, 0--0.70; P = 0.020, and RH = 0; 95% CI, 0--0.71; P = 0.021, respectively). When examined before 5 years of age (n = 81), the children with CCR5 wt/Delta 32 had significantly lower levels of cell-associated HIV-1 DNA than wild-type homozygotes (P = 0.016, adjusted by race), while SDF1-3'A carriers had relatively higher levels (P = 0.047, adjusted by race). Although the disease-retarding effect of CCR5 wt/Delta 32 subsequently disappeared, time to death was still significantly delayed in the CCR5 Delta 32 heterozygotes without SDF1-3'A (RH = 0; 95% CI, 0--0.53; P = 0.008). CONCLUSION: In pediatric AIDS, the protective effect of CCR5 wt/Delta 32 is more pronounced in early years of infection and appears to be abrogated by the SDF1-3'A genotype.
Subject(s)
Chemokines, CXC/genetics , HIV Infections/genetics , HIV-1 , Receptors, CCR5/genetics , Adolescent , Alleles , Base Sequence , Chemokine CXCL12 , Child , Child, Preschool , DNA, Viral/blood , Disease Progression , Genotype , HIV Infections/epidemiology , HIV Infections/pathology , Heterozygote , Humans , Infant , Proportional Hazards Models , Sequence Deletion , Survival AnalysisABSTRACT
Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2). A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.
Subject(s)
Anti-HIV Agents/pharmacology , Fusion Proteins, gag-pol/genetics , Gene Targeting , HIV-1/drug effects , Peptide Nucleic Acids/pharmacology , Virus Replication/drug effects , 3' Untranslated Regions/genetics , Base Sequence , Cell Line , Cells, Cultured , Enzyme Activation , Fusion Proteins, gag-pol/metabolism , Gene Products, gag/biosynthesis , Gene Products, gag/metabolism , HIV Core Protein p24/biosynthesis , HIV Infections/virology , HIV Protease/biosynthesis , HIV Protease/chemistry , HIV Protease/genetics , HIV Protease/metabolism , HIV-1/genetics , HIV-1/physiology , Humans , Molecular Sequence Data , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/metabolism , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Virion/physiology , gag Gene Products, Human Immunodeficiency Virus , pol Gene Products, Human Immunodeficiency VirusABSTRACT
Population genetic study of the complicated STR locus-FGA was performed by the method of PCR in Han population samples. A total of 21 different alleles including 7 interalleles and 63 different genotypes were observed in 349 unrelated individuals. The observed genotype discrimination showed no significant deviation from Hardy-Weinberg equilibrium and 51 family studies showed no mutation. According to the results obtained in this study(DP = 0.9612, PE = 0.7007, PIC = 0.8333), this system can be used as an useful means in forensic identification of both criminal and paternity case.
Subject(s)
Genetics, Population , Polymorphism, Genetic , Tandem Repeat Sequences , Asian People/genetics , China , Fibrinogen/genetics , Gene Frequency , Genotype , Humans , Polymerase Chain ReactionABSTRACT
Using the primers designed by Yoshida, the amplified fragment length polymorphism of the STR locus CSF1PO was studied. 8 alleles were determined from 312 unrelated individuals of Han population living in Wuhan. Sensitivity and suitability of the PCR-STR typing using Yoshida's primers were compared with the one using Hammond's. Results proved that Yoshida's method were more suitable in forensic identification than that of Hammond, especially in the typing of severely degraded DNA samples.
