Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue

Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.     

Fermentation of galacturonic acid and pectin-rich materials to ethanol by genetically modified strains of Erwinia

Evaluation of the four ethanologenic constructs of bacteria in the genus Erwinia indicates that two strains E. chrysanthemi EC16 and E. carotovora SR38 show promise for development of direct hydrolysis and fermentation of pectin-rich substrates to mixtures of ethanol and acetate. Both strains fermented glucose to ethanol in nearly theoretical yields, but produced mainly acetate and ethanol by fermentation of D-galacturonic acid. Both strains depolymerized citrus pectin, polygalacturonic acid and polysaccharides in citrus peel and converted resulting sugars to carbon dioxide, acetate, ethanol and lesser amounts of formate and succinate.     

Probing the stability of the SpCas9–DNA complex after cleavage

CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region.

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro. Received: 12 October 1998 / Accepted: 28 February 1999     

IFN-gamma inhibits presentation of a tumor/self peptide by CD8 alpha- dendritic cells via potentiation of the CD8 alpha+ subset

Using an in vivo model of tumor/self peptide presentation for induction of class I-restricted skin test reactivity, we have previously shown that a minority population of CD8+ dendritic cells (DC) negatively regulates the induction of T cell reactivity by peptide-loaded CD8- DC in DBA/2 mice. However, the CD8- fraction can be primed by IL-12 to overcome inhibition by the CD8+ subset when the two types of DC are cotransferred into recipient hosts. We report here that exposure of CD8+ DC to IFN-gamma greatly enhances their inhibitory activity on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8- DC to overcome suppression. In contrast, IFN-gamma has no direct effects on the APC function of the latter cells and does not interfere with IL-12 signaling. The negative regulatory effect triggered by IFN-gamma in CD8+ DC appears to involve interference with tryptophan metabolism in vivo. Through tryptophan depletion affecting T cell responses, IFN-gamma acting on CD8+ DC may thus contribute to regulation of immunity to tumor/self peptides presented by the CD8- subset.     

IL-12 induces SDS-stable class II alphabeta dimers in murine dendritic cells

We assessed the effect of rIL-12 on the expression of class II molecules and on the ratio between SDS-stable and unstable alphabeta dimers in dendritic cells. We found that in vitro exposure of the cells to IL-12 increased their surface expression of mature class II molecules, despite a marked decline in class II biosynthesis. This effect was accompanied by a striking increase in the overall proportion of SDS-stable heterodimers.     

Site-specific differences of insulin action in adipose tissue derived from normal prepubertal children

Body fat distribution determines obesity-related morbidity in adults but little is known of the aetiology or pathophysiology in children. This study investigates differences in insulin-mediated metabolism in primary cell cultures of subcutaneous and visceral preadipocytes derived from prepubertal children. The impact of differentiation and responses to TNFalpha exposure was also investigated. Proliferation rates were greater in subcutaneous versus visceral preadipocytes (41 h3 versus 69 h4; P=0.008). Insulin caused a dose-dependent increase in GSK-3 phosphorylation and an increase in MAPK phosphorylation over time, with increased sensitivity in subcutaneous preadipocytes. Post-differentiation, dose-dependent increases in GSK-3 phosphorylation were maintained, while MAPK phosphorylation was identical in both subtypes. No changes were observed in insulin receptor abundance pre-/post-differentiation. GLUT4 abundance was significantly increased in visceral versus subcutaneous adipocytes by 76(4)%; P=0.03), coincidental with increased insulin-stimulated 2-deoxy-glucose transport (+150(26)% versus +79(10)%; P=0.014) and further elevated by acute exposure to TNFalpha (+230(52)%; P=0.019 versus +123(24)%; P=0.025, respectively). TNFalpha also significantly increased basal glucose transport rates (+44(14)%; P=0.006 versus +34(11)%; P=0.007) and GLUT1 localisation to the plasma membrane. These data establish site-specific differences in subcutaneous and visceral fat cells from children. Responses to insulin varied with differentiation and TNFalpha exposure in the two depots, consistent with parallel changes in GLUT1/4 abundance and localisation.     

CD40 ligation ablates the tolerogenic potential of lymphoid dendritic cells

The outcome of dendritic cell (DC) presentation of P815AB, a tolerogenic tumor/self peptide, depends on a balance between the respective immunogenic and tolerogenic properties of myeloid (CD8 alpha(-)) and lymphoid (CD8 alpha(+)) DC. We have previously shown that CD8(-) DC can be primed by IL-12 to overcome inhibition by the CD8(+) subset and initiate immunogenic presentation in vivo when the two types of peptide-pulsed DC are cotransferred into recipient hosts. IFN-gamma enhances the inhibitory activity of CD8(+) DC on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8(-) DC to overcome suppression. We report here that CD40 ligation on lymphoid DC ablated their inhibitory function on Ag presentation as well as IFN-gamma potentiation of the effect. CD40 modulation of IFN-gamma action on lymphoid DC involved a reduction in IFN-gamma R expression and tryptophan-degrading ability. This effect was accompanied in vitro by an impaired capacity of the CD40-modulated and IFN-gamma-treated DC to initiate T cell apoptosis. In vivo, not only did CD40 triggering on lymphoid DC abrogate their tolerogenic activity, but it also induced the potential for immunogenic presentation of P815AB. Importantly, a pattern similar to P815AB as well as CD40 modulation of lymphoid DC function were observed on testing reactivity to NRP, a synthetic peptide mimotope recognized by diabetogenic CD8(+) T cells in nonobese diabetic mice.