Evaluation of InnoTyper® 21 in a sample of Rio de Janeiro population as an alternative forensic panel.

The use of bi-allelic markers such as retrotransposable element insertion polymorphisms or Innuls (for insertion/null) can overcome some limitations of short tandem repeat (STR) loci in typing forensic biological evidence. This study investigated the efficiency of the InnoTyper® 21 Innul markers in an urban admixed population sample in Rio de Janeiro (n = 40) and one highly compromised sample collected as evidence by the Rio de Janeiro police. No significant departures from Hardy-Weinberg equilibrium were detected after the Bonferroni correction (α' ≈ 0.05/20, p < 0.0025), and no significant linkage disequilibrium was observed between markers. Assuming loci independence, the cumulative random match probability (RMP) was 2.3 × 10-8. A lower mean Fis value was obtained for this sample population compared with those of three North American populations (African-American, Southwest Hispanic, US Caucasian). Principal component analysis with the three North American populations and one from 21 East Asian population showed that African Americans segregated as an independent group while US Caucasian, Southwest Hispanic, East Asian, and Rio de Janeiro populations are in a single large heterogeneous group. Also, a full Innuls profile was produced from an evidence sample, despite the DNA being highly degraded. In conclusion, this system is a useful complement to standard STR kits.

Biological treatment in systemic juvenile idiopathic arthritis: achievement of inactive disease or clinical remission on a first, second or third biological agent.

OBJECTIVES: To analyse the effect of biological agents (BAs) in terms of achieving inactive disease (ID) or clinical remission (CR) in patients with systemic juvenile idiopathic arthritis (SJIA), to describe effects of switching or discontinuing a BA and to assess the proportion of patients able to maintain ID or CR off steroids and after withdrawing BA therapy. METHODS: Retrospective study in a French paediatric rheumatology reference centre using the CEMARA (CEntre des MAladies RAres) register. RESULTS: Seventy-seven patients were included with a cumulative follow-up of 245.5 patient-years (median 1.1, range 0.5-8.0). On a first BA, ID was achieved in 37 patients, including 1 patient out of 12 patients on etanercept, 26 patients out of 51 on anakinra and 7 out of 10 on canakinumab. One patient on abatacept and two patients on tocilizumab also achieved ID. Switching of BA was common. The switch to a second (n=34), third (n=18) or fourth (n=4) BA resulted in ID in a further 13 patients, either on canakinumab (n=6) or tocilizumab (n=7). At last follow-up, 40 patients were in CR (27 patients off steroids, 5 patients having never received steroid treatment), either on (n=29) or off (n=11) BA. CONCLUSIONS: In this series of patients with SJIA, interleukin-1 inhibitors were associated with a higher proportion of ID than tumour necrosis factor inhibitors when used as first BA. Switching allowed some patients to achieve ID when treated with canakinumab or tocilizumab. CR was eventually achieved in more than half of the patients.

MicroRNA-206 functions as a pleiotropic modulator of cell proliferation, invasion and lymphangiogenesis in pancreatic adenocarcinoma by targeting ANXA2 and KRAS genes.

Recent advances in cancer biology have emerged important roles for microRNAs (miRNAs) in regulating tumor responses. However, their function in mediating intercellular communication within the tumor microenvironment is thus far poorly explored. Here, we found miR-206 to be abrogated in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. We show that miR-206 directly targets the oncogenes KRAS and annexin a2 (ANXA2), thereby acting as tumor suppressor in PDAC cells by blocking cell cycle progression, cell proliferation, migration and invasion. Importantly, we identified miR-206 as a negative regulator of oncogenic KRAS-induced nuclear factor-κB transcriptional activity, resulting in a concomitant reduction of the expression and secretion of pro-angiogenic and pro-inflammatory factors including the cytokine interleukin-8, the chemokines (C-X-C motif) ligand 1 and (C-C motif) ligand 2, and the granulocyte macrophage colony-stimulating factor. We further show that miR-206 abrogates the expression and secretion of the potent pro-lymphangiogenic factor vascular endothelial growth factor C in pancreatic cancer cells through an NF-κB-independent mechanism. By using in vitro and in vivo approaches, we reveal that re-expression of miR-206 in PDAC cells is sufficient to inhibit tumor blood and lymphatic vessel formation, thus leading to a significant delay of tumor growth and progression. Taken together, our study sheds light onto the role of miR-206 as a pleiotropic modulator of different hallmarks of cancer, and as such raising the intriguing possibility that miR-206 may be an attractive candidate for miRNA-based anticancer therapies.

Expression of c-Fos in Alko alcohol rats responding for ethanol in an operant paradigm.

BACKGROUND: Identification of the brain regions involved in ethanol administration is important for understanding the neurobiology of ethanol addiction. Animal studies with different brain mapping techniques found that voluntary ethanol self-administration leads to changes in activity of specific brain regions in patterns that only partially overlap with patterns of brain regions affected by involuntary (i.e., experimenter-administered) ethanol administration. As an extension of studies mapping changes in neural activity after voluntary ethanol drinking, this study analyzed expression of the inducible transcription factor c-Fos after ethanol consumption in an operant procedure. METHODS: AA (Alko alcohol) rats were trained to operantly respond for water, 0.2% saccharin, 0.2% saccharin/10% (w/v) ethanol, or 10% ethanol in a 30-min limited-access procedure. Animals were allowed to self-administer solutions for at least 40 ethanol response sessions and were killed 1.5 hr after beginning of the last session. Forty-seven brain regions were immunohistochemically analyzed for c-Fos expression. RESULTS: In this paradigm, ethanol dose-dependently increased c-Fos expression in the Edinger-Westphal nucleus (EW) and decreased expression in the dorsal tenia tecta compared with no-ethanol controls. No effects of saccharin on c-Fos expression were found. CONCLUSIONS: Our results extend previous findings of preferential sensitivity of EW to alcohol in voluntary self-administration procedures to operant responding for ethanol and warrant further investigation of ethanol's effects on the EW. The finding that ethanol attenuated c-Fos expression in the tenia tecta is novel. Taken together, these findings confirm that voluntary ethanol self-administration leads to changes in activity of a limited number of brain regions with previously unexamined roles in ethanol sensitivity and addiction.

Characterization of the initiator and downstream promoter elements of herpes simplex virus 1 late genes.

Previously identified cis-acting regulatory elements of herpes simplex virus (HSV) 1 late promoters include a TATA element upstream from the start of transcription, an initiator-like element at the start of transcription, and sequences downstream from the start of transcription. To determine whether these elements are functionally equivalent to similar elements from other eukaryotic genes, model late promoters were constructed using well-characterized regulatory elements from non-HSV genes. These modular promoters were then inserted into the viral genome upstream from a lacZ marker gene. Results showed that a eukaryotic initiator element, along with a TATA element, can function as a late HSV promoter. Several initiator sequences from both viral and nonviral genes were functionally similar to the initiator-like element in HSV-1 late promoters; however, a random sequence of the same size and a similarly located sequence from the HSV-1 early thymidine kinase promoter could not substitute for the initiator element. These results indicate that eukaryotic initiator elements are functionally equivalent to HSV-1 late promoter initiator elements. In addition, the downstream element of the late glycoprotein C promoter was further analyzed by construction of a series of small deletions and insertions. The presence of the downstream glycoprotein C region in a promoter consisting of a strong TATA and initiator element increased mRNA expression by a modest amount; this effect appeared to be sequence specific and dependent on its exact alignment with the upstream elements of the promoter.

Generation of HIV-1/HIV-2 cross-reactive peptide antisera by small sequence changes in HIV-1 reverse transcriptase and integrase immunizing peptides.

We have generated peptide antisera against selected regions in HIV-1 and HIV-2 reverse transcriptase (RT) and integrase (IN) to investigate the specificity of determinants governing the immune response. Peptides representing homologous regions (>50%) in the N- and C-termini and central portions of these proteins were synthesized and injected into rabbits. HIV-1 and HIV-2 IN peptide antisera inhibited IN-mediated cleavage of an HIV-1 DNA oligonucleotide substrate in a 3' processing assay, while anti-RT or normal sera had no effect. None of the RT sera inhibited RT activity. In Western blots, HIV-2 antisera directed against RT or IN peptides recognized HIV-2 RT and IN proteins, respectively, as expected, but also cross-reacted with the corresponding HIV-1 proteins. By contrast, corresponding HIV-1 antisera were type-specific. In some cases, HIV-1 cross-reactive antisera could be generated by immunization with HIV-1 chimeric peptides with as few as two residues in the HIV-1 sequence changed to the corresponding HIV-2 amino acids. The finding that a type-specific response can be converted to a cross-reactive response suggests alternate strategies for developing new diagnostic reagents which detect HIV-1 and HIV-2. In addition, our results provide a general model for generating HIV peptide vaccines with dual specificity against HIV-1 and HIV-2.

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.

Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.

Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Epitope mapping of the HIV-1 gag region by analysis of gag gene deletion fragments expressed in Escherichia coli defines eight antigenic determinants.

Immune response to HIV infection is generally characterized by appearance of antibodies to the gag protein p24 early in infection, and by apparent loss of p24 antibodies accompanied by increases in p24 antigen levels with disease progression. Precise definition of the immunodominant epitopes present in gag gene proteins has potential clinical significance. Seventeen anti-gag monoclonal antibodies (MAb) were used in enzyme-linked immunosorbent assays (ELISA) with antigens expressed by nine recombinant clones to define epitopes on HIV gag proteins which elicit an immune response. All of the MAbs tested, except two anti-p17, reacted with a clone which expresses the carboxyl terminal 13 amino acids of p17 and all of p24 and p15. All anti-p24 MAbs reacted with clones containing all of p24. MAbs reacted differentially with clones containing deleted regions depending on the antigenic portion expressed. Of thirteen potential identifiably different genomic regions which could be predicted from the genomic structure of the clones, eight different antigen epitopes were defined: two on p17, five on p24, and one on p15 (in the region corresponding to the carboxyl terminal protein p6). Six regions did not appear to react with any of the monoclonal antibodies available. Identification of the epitopes present in the cloned antigens should allow their use to evaluate sera from HIV-infected donors at different clinical stages of progression to AIDS.

Reactivity of an HIV gag gene polypeptide expressed in E. coli with sera from AIDS patients and monoclonal antibodies to gag.

A segment of the gag gene of the human immunodeficiency virus (HIV) (HTLV-IIIB strain), the virus which causes acquired immunodeficiency syndrome (AIDS), has been cloned into the bacterial expression vector, pCQV2, and mapped to the right-hand portion of the gag gene containing the carboxyl-terminal portion of p24 and the amino-terminal portion of p15. Nucleic-acid sequencing of the insert-vector junctions further defined the 5'-terminal nucleotide of HIV sequence as nucleotide 997 and the 3'-terminal nucleotide as 1696. When used in an enzyme-linked immunosorbent assay (ELISA) with sera from HIV-infected patients, the cloned antigen reacted with a subset of sera which were positive on a standard ELISA using whole virus as antigen. Western-blot screening of these sera with whole virus indicated that all p24-positive sera were positive with the clone, suggesting that the carboxyl-terminal portion of p24 contains a highly antigenic epitope(s). A serum which was p24-negative p15-positive by Western blot analysis was also highly reactive, indicating that a p15 epitope is present in the cloned antigen. Epitope mapping with a series of monoclonal antibodies to gag resulted in positive ELISA with 2 of 3 anti-p24, 0 of 1 anti-p15, and 0 of 1 anti-p17 Western-blot-positive monoclonal antibodies, suggesting that one of the anti-p24 monoclonal antibodies reacts with epitopes amino-terminal to those coded from nucleotide 997, two anti-p24 monoclonals react with epitopes carboxyl-terminal to those coded from nucleotide 997, and the anti-p15 monoclonal reacts with epitopes carboxyl-terminal to those coded from nucleotide 1696.

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages.

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.