Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

Aptamers: new arrows to target dendritic cells.

Aptamers, as a novel class of molecular probes for diagnosis, imaging and targeting therapy, have attracted increasing attention in recent years. Aptamers are generated from libraries of single-stranded nucleic acids against different molecules via the "systematic evolution of ligands by exponential enrichment" (SELEX) method. SELEX is a repetitive process of a sequential selection procedure in which a DNA or RNA library pool is incubated separately with target and control molecules to select specific oligonucleotide aptamers with high affinities and specificities. Cell-SELEX is a modified version of the SELEX process in which whole living cells are used as targets for the aptamers. Dendritic cell (DC) targeting, as a new therapeutic approach, can improve the efficiency of immunotherapy in the treatment of allergies and cancers. DCs use various receptors to continuously induce adaptive immunity via capture and presentation of antigens to naïve T cells. DCs are considered as the best targets in modulating immune responses against cancer, autoimmunity, allergy and transplantation. Aptamers, as a new agent, can be applied in DC targeting. The purpose of this review is to present some general concepts of aptamer production and DC targeting by aptamer molecules.

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran.

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged < or = 12 years in the primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 before (1985-2000) to 0.77 per 1000 child population after the intervention (2001-07). In a control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests and timely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas.

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged ≤ 12 years inthe primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic ofIran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referredfor physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88before (1985–2000) to 0.77 per 1000 child population after the intervention (2001–07). In a control area with nosurveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests andtimely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas

En 2001, un système de surveillance de la leishmaniose viscérale a été mis en place pour les enfants âgésde 0 à 12 ans dans le système de santé primaire du district de Meshkin-Shahr, province d’Ardebil, nord-ouest dela République islamique d’Iran. Tous les cas présentant des signes cliniques et des symptômes de leishmanioseviscérale ainsi qu’une réaction positive au test d’agglutination directe étaient orientés en vue d’un examen physiqueet d’un traitement. L’incidence annuelle moyenne de la leishmaniose viscérale a nettement diminué, passant de1,88 avant l’intervention (1985-2000) à 0,77 pour 1 000 enfants après l’intervention (2001-2006). Elle a augmentédans une zone témoin sans surveillance, passant de 0,11 à 0,23 pour 1 000 enfants. Un dépistage précoce dela leishmaniose viscérale à l’aide de tests sérologiques pratiques et une prise en charge rapide des cas permettraientde réduire les taux de mortalité et de morbidité de la leishmaniose viscérale dans les zones endémiques

Correlation of the frequency of petite formation by isolates of Saccharomyces cerevisiae with virulence.

In previous studies on the colony phenotype switching of Saccharomyces cerevisiae, we observed that the least virulent isolates formed greater numbers of petite colonies when grown at body temperature, 37 degrees C. To determine if there is a link between virulence and petite formation, we examined the frequency of spontaneous petite formation for virulent clinical isolates (YJM128, YJM309), an intermediate virulent segregant of YJM128 (YJM145) and avirulent clinical (YJM308) and nonclinical S. cerevisiae (Y55, YJM237) after growth at 37 degrees C. The rank order of increasing frequency of petite formation was YJM128 = YJM145 < YJM309 < Y 55 < YJM308 = YJM237, which is similar to the rank-order of virulence in CD-1 mice. To assess the virulence of petites in vivo, two mouse models, CD-1 and DBA/ 2N, were infected i.v. with 10(7) cfu of either the parental grand or a spontaneously derived petite from one of four isolates previously classified with differing degrees of virulence: YJM128, YJM309, YJM145 and Y55. In both CD-1 and DBA/2N, the mean log10 cfu of grands recovered from the brain was significantly higher than that of the petites (P<0001). Overall, petites were significantly less virulent than the parental strains. However, death of some DBA/2N mice caused by YJM128 petite 1 showed that petites are not totally avirulent. To see if S. cerevisiae isolates form petite colonies in vivo, both mouse models were infected with parental grands of YJM128 and Y55. Recovered colonies were counted and confirmed as grand or petite, and the frequency of petite colonies in the brain, the target organ, correlated with the in vitro results. Overall, these studies show an inverse correlation between the frequency of petite-colony formation and the previously determined virulence of S. cerevisiae in CD-1 mice. Furthermore, petites were significantly less virulent than the parental grands, in most cases, and petites are spontaneously formed in vivo at a frequency inversely correlated to the virulence of the strain.

Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

Syn-capping of human T lymphocyte adhesion/activation molecules and their redistribution during interaction with endothelial cells.

Lymphocyte-endothelial cell interactions are mediated in part by multiple lymphocyte surface adhesion/activation molecules and their cognate ligands. We investigated the surface localization of several of these molecules implicated in T cell adhesion and transendothelial migration mechanisms to determine if spatial regulation of their distribution contributes to these processes. T lymphocyte suspensions were stained to define distribution, ability to be aggregated into energy-dependent caps, and potential cocapping of several adhesion structures. CD2, CD44, L-selectin (LAM-1, LECCAM-1), and CD11a/CD18 (LFA-1) exhibited uniform distribution on the T cell surface by direct immunofluorescence but formed caps in an energy-dependent, and therefore cytoskeletally driven, manner when examined by indirect immunofluorescence. CD2 was shown to syn-cap (unidirectionally cocap) with CD44 and CD11a/CD18 (LFA-1), an observation potentially related to functional cooperation among these molecules in T cell activation. T cells were also added to endothelial cell monolayers to assess, in a physiologically relevant context, potential surface molecule reorganization. Lymphocytes co-cultured with human umbilical vein endothelial cells (HUVEC) underwent a profound shape change, from essentially round cells to polarized cells bearing pseudopodia. Immunofluorescent localization of T cell adhesion/activation molecules using confocal microscopy revealed the redistribution of CD2, CD44, and L-selectin to the pseudopod. In contrast, CD11a/CD18 remained globally distributed on the cell surface, even in severely deformed cells. Both lymphocyte shape change and membrane molecule redistribution appear to be cell-cell contact-dependent phenomena requiring intact, viable endothelial cells. Mechanisms that control these events may be critical to lymphocyte recirculation and inflammation.