25 research outputs found
Analysis of temporal expression of HTLV-2 reveals similarities and functional differences from HTLV-1
In the present study, we developed a robust splice site-specific real-time RT-PCR method to quantitate all HTLV-2 transcripts. Results of this analysis conducted on three different infected cell lines (HTLV-2A Mo-T , C344 and HTLV-2B BJAB-Gu) showed that the most abundant mRNA was gag/pol followed by the accessory transcript 1-3, coding for the p28 and for p22/p20 proteins. The third most abundant mRNA was tax/rex.
To investigate if different mRNAs produced by HTLV-2 are expressed at different levels upon viral reactivation, we studied the kinetics of viral expression in PBMCs from three subjects infected with HTLV-2B and cultured in vitro for 48 hours. The level of expression of the full length gag/pol transcript was the highest in all samples. The tax/rex mRNA was detected already at time zero and increased very rapidly following in vitro culture, reaching the highest copy number between zero and 2-4 hours. The minus-strand APH-2 mRNA, was expressed at high level. As observed in the infected cell lines, the 1-3 mRNA was expressed at high levels in all subjects. This finding is particularly intriguing, as it encodes two proteins that were shown to exert a powerful control on Tax and Rex function. This peculiar pattern of expression, which is in striking contrast with that of HTLV-1, might in part explain the differential pathogenicity of the two viruses
Kinetics of human-T cell leukemia virus type 2 (HTLV-2) mRNA expression in PBMCs isolated from infected subjects
Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities
Background: Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. the possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal Findings: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. the inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/Significance: the high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. the easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.Department of Education, Universities and Research, Basque GovermentFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Istituto Superiore di Sanita, National Research Project on A.I.D.S.Cariparma Banking FoundationBrazilian National Research CouncilUniv Parma, Sez Microbiol, Dipartimento Patol, I-43100 Parma, ItalyUniv Basque Country, Fac Med Odontol, Dept Inmunol, Microbiol Parasitol, Bilbao, SpainUniv Basque Country, Dept Enfermeria I, Bilbao, SpainUniv Milan, Dipartimento Sci Cliniche L Sacco, Sez Malattie Infettive Immunopatol, Milan, ItalyUniv Studi Parma, Dipartimento Clin Med, Nefrol Sci Prev, Parma, ItalyUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilDepartment of Education, Universities and Research, Basque Goverment: IT-264-07FAPESP: 06/50634-2Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 50G.30Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 40D.14Cariparma Banking Foundation: 2004.0190Brazilian National Research Council: research fellowshipWeb of Scienc
Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence for distinct expression kinetics with nuclear accumulation of APH-2 mRNA
Background: Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are delta retroviruses with similar genetic organization. Although both viruses immortalize T-cells in vitro, they exhibit distinct pathogenic potential in vivo. To search for possible differences in its expression strategy with respect to HTLV-1, we investigated the pattern of HTLV-2 expression in infected cell lines and peripheral blood mononuclear cells (PBMCs) from infected patients using splice site-specific quantitative RT-PCR.
Findings: A novel alternative splice acceptor site for exon 2 was identified; its usage in env transcripts was found to be subtype-specific. Time-course analysis revealed a two-phase expression kinetics in an infected cell line and in PBMCs of two of the three patients examined; this pattern was reminiscent of HTLV-1. In addition, the minus-strand APH2 transcript was mainly detected in the nucleus, a feature that was similar to its HTLV-1 orthologue HBZ. In contrast to HTLV-1, expression of the mRNA encoding the main regulatory proteins Tax and Rex and that of the mRNAs encoding the p28 and truncated Rex inhibitors is skewed towards p28/truncated Rex inhibitors in HTLV-2.
Conclusion: Our data suggest a general converging pattern of expression of HTLV-2 and HTLV-1 and highlight peculiar differences in the expression of regulatory proteins that might influence the pathobiology of these viruse
Vaccination of Lactating Dairy Cows for the Prevention of Aflatoxin B-1 Carry Over in Milk
The potential of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB1-KLH) in
controlling the carry over of the aflatoxin B1 (AFB1) metabolite aflatoxin M1 (AFM1) in cow milk is reported. AFB1 is the most
carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB1 is AFB1 chemically modified as AFB1-1(Ocarboxymethyl)
oxime. In comparison to AFB1, AnAFB1 has proven to be non-toxic in vitro to human hepatocarcinoma cells
and non mutagenic to Salmonella typhimurium strains. AnAFB1-KLH was used for immunization of cows proving to induce a
long lasting titer of anti-AFB1 IgG antibodies (Abs) which were cross reactive with AFB1, AFG1, and AFG2. The elicited anti-
AFB1 Abs were able to hinder the secretion of AFM1 into the milk of cows continuously fed with AFB1. Vaccination of
lactating animals with conjugated AnAFB1 may represent a solution to the public hazard constituted by milk and cheese
contaminate
Average AFM<sub>1</sub> concentrations in milk collected during the second experimental period from high responder cows.
<p>Three high responder vaccinated cows (♦) and six control cows (▴) were fed 145 µg of AFB<sub>1</sub>/day from day 1 to day 11. Data are presented as mean ± SD.</p
In vitro effects of AFB<sub>1</sub> (A) and AnAFB<sub>1</sub> (B) on human hepatoblastoma cell viability.
<p>In vitro effects of AFB<sub>1</sub> (A) and AnAFB<sub>1</sub> (B) on human hepatoblastoma cell viability.</p
Mutagenicity of AFB<sub>1</sub> and AnAFB<sub>1</sub> in <i>S. typhimurium</i> TA98 and TA100.
<p>*mean of two independent, triplicate mutagenicity assays with SD.</p
Titers of anti-AFB<sub>1</sub> Abs.
<p>Cows numbered 322, 335, 338, 348, 363 and 366 were initially <i>i.m.</i> primed with 500 µg of AnAFB<sub>1</sub>-KLH conjugate and then boosted at week 3, 6 and 9 with the same amount of immunogen. Ab titers (presented in figure on a logarithmic scale) were determined by the method described in the text.</p
Average AFM<sub>1</sub> concentrations in milk collected during the first experimental period from high responder cows.
<p>Three high responder vaccinated cows (♦) and six control cows (▴) were fed 144 µg of AFB<sub>1</sub>/day from day 1 to day 11. Data are presented as mean ± SD.</p