Antagonistic Interplay between MicroRNA-155 and IL-10 during Lyme Carditis and Arthritis

MicroRNA-155 has been shown to play a role in immune activation and inflammation, and is suppressed by IL-10, an important anti-inflammatory cytokine. The established involvement of IL-10 in the murine model of Borrelia burgdorferi-induced Lyme arthritis and carditis allowed us to assess the interplay between IL-10 and miR-155 in vivo. As reported previously, Mir155 was highly upregulated in joints from infected severely arthritic B6 Il10^-/- mice, but not in mildly arthritic B6 mice. In infected hearts, Mir155 was upregulated in both strains, suggesting a role of miR-155 in Lyme carditis. Using B. burgdorferi-infected B6, Mir155^-/-, Il10^-/-, and Mir155^-/- Il10^-/- double-knockout (DKO) mice, we found that anti-inflammatory IL-10 and pro-inflammatory miR-155 have opposite and somewhat compensatory effects on myeloid cell activity, cytokine production, and antibody response. Both IL-10 and miR-155 were required for suppression of Lyme carditis. Infected Mir155^-/- mice developed moderate/severe carditis, had higher B. burgdorferi numbers, and had reduced Th1 cytokine expression in hearts. In contrast, while Il10^-/- and DKO mice also developed severe carditis, hearts had reduced bacterial numbers and elevated Th1 and innate cytokine expression. Surprisingly, miR-155 had little effect on Lyme arthritis. These results show that antagonistic interplay between IL-10 and miR-155 is required to balance host defense and immune activation in vivo, and this balance is particularly important for suppression of Lyme carditis. These results also highlight tissue-specific differences in Lyme arthritis and carditis pathogenesis, and reveal the importance of IL-10-mediated regulation of miR-155 in maintaining healthy immunity.     

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on 4-hydroxy-2-nonenal-induced apoptotic T cell death

4-Hydroxy-2-nonenal (HNE), the aldehydic product of lipid peroxidation, is associated with multiple immune dysfunctions, such as HIV and hepatitis C virus infection. HNE-induced immunosuppression could be due to a decrease in CD4+ T lymphocyte activation or proliferation. Glutathione (GSH) is the most abundant endogenous antioxidant in cells, and an adduct between HNE and GSH has been suggested to be a marker of oxidative stress. Our earlier studies showed that HNE induced cytotoxicity and Akt inactivation, which led to the enhancement of FasL expression and concomitantly decreased cellular FLICE-like inhibitory protein (c-FLIP(S)) levels. In this study, we found that HNE caused intracellular GSH depletion in Jurkat T cells, and we further investigated the role of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA), a GSH prodrug, in attenuating HNE-induced cytotoxicity in CD4+ T lymphocytes. The results show that PTCA protected against HNE-induced apoptosis and depletion of intracellular GSH. PTCA also suppressed FasL expression through increasing levels of Akt kinase as well as antiapoptotic c-FLIP(S) and decreasing the activation of type 2 protein serine/threonine phosphatase. Taken together, these data demonstrate a novel correlation between GSH levels and Akt activation in T lymphocyte survival, which involves FasL down-regulation and c-FLIP(S) expression through increasing intracellular GSH levels. This suggests that PTCA could potentially be used in the treatment of oxidative stress-induced immunosuppressive diseases.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

The radial forearm skin graft-fascial flap

The radial forearm flap has become a versatile flap for upper extremity reconstruction. The use of the forearm flap for hand reconstruction in the patient with previously burned forearms has not been widely appreciated. In those patients whose forearms have been previously split-thickness skin-grafted on fascia, we have employed the reverse radial forearm flap as a skin graft-fascial flap for hand reconstruction and have obtained excellent functional results. Three patients at various intervals postburn are presented to demonstrate use of this flap for wrist contracture release, coverage of arthroplasties, first web space contracture release, and acute salvage of phalanges and tendons. Assessment of the hand's vascular anatomy and careful treatment of the donor area have contributed to no added morbidity and an excellent aesthetic result at the donor site.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains

Background  

Clostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.     

Peptide-functionalized poly(ethylene glycol) star polymers: DNA delivery vehicles with multivalent molecular architecture

Exploring the development of nonviral nucleic acid delivery vectors with progressive, specific, and novel designs in molecular architecture is a fundamental way to investigate how aspects of chemical and physical structure impact the transfection process. In this study, macromolecules comprised of a four-arm star poly(ethylene glycol) and termini modified with one of five different heparin binding peptides have been investigated for their ability to bind, compact, and deliver DNA to mammalian cells in vitro. These new delivery vectors combine a PEG-derived stabilizing moiety with peptides that exhibit unique cell-surface binding ability in a molecular architecture that permits multivalent presentation of the cationic peptides. Five peptide sequences of varying heparin binding affinity were studied; each was found to sufficiently bind heparin for biological application. Additionally, the macromolecules were able to bind and compact DNA into particles of proper size for endocytosis. In biological studies, the PEG-star peptides displayed a range of toxicity and transfection efficiency dependent on the peptide identity. The vectors equipped with peptides of highest heparin binding affinity were found to bind DNA tightly, increase levels of cellular internalization, and display the most promising transfection qualities. Our results suggest heparin binding peptides with specific sequences hold more potential than nonspecific cationic polymers to optimize transfection efficiency while maintaining cell viability. Furthermore, the built-in multivalency of these macromolecules may allow simultaneous binding of both DNA at the core of the polyplex and heparan sulfate on the surface of the cell. This scheme may facilitate a bridging transport mechanism, tethering DNA to the surface of the cell and subsequently ushering therapeutic nucleic acids into the cell. This multivalent star shape is therefore a promising architectural feature that may be exploited in the design of future polycationic gene delivery vectors.