Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8a(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8a(+) DCs are sufficient to subsequent CD8(+) T cell responses.

Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors

Background: Small molecule inhibitors of the zinc finger domain (ZFI) in the nucleocapsid protein (NCp7) of HIV-1 are potent inhibitors of HIV and SIV
replication and may have utility as topical products to prevent infection. Furthermore, intravaginal rings (IVRs) were developed as coitally-independent,
sustained release devices which could be used for administration of HIV microbicides. The aims of these studies were to demonstrate that IVRs sized for
macaques are practical and compatible with the current generation of thioester-based NCp7 inhibitors.

Methods: Non-medicated silicone elastomer vaginal rings of various sizes thought to be applicable for macaques were prepared and tested for vaginal fit in Pigtailed and Chinese Rhesus macaques. Macaques were monitored for 8 weeks for mucosal disruption by colposcopy and proinflammatory cytokine markers in cervical vaginal lavages (CVL) using Luminex bead-based technology. Three different ZFIs (compounds 52, 89 and 122, each derived from an N-substituted S-acyl-2-mercaptobenzamide thioester scaffold) were loaded at 50 mg into an optimal matrix-type ring design. In vitro continuous release studies were then conducted over 28 days and analyzed by HPLC. Rate of release was determined by linear regression analysis.

Results: Qualitative evaluation at the time of ring insertion suggested that the 25 mm ring provided optimal fit in both macaque species. All rings remained in
place during the study period (2 to 4 weeks), and the animals did not attempt to remove the rings. No tissue irritation was observed, and no signs of physical
discomfort were noted. Also, no significant induction of cervicovaginal proinflammatory markers was observed during the 8-week period during and following ring insertion. One Pigtailed macaque showed elevated IL-8 levels in the CVL during the period when the ring was in place; however, these levels were comparable to those observed in two control macaques. In vitro release of the ZFIs peaked at day 1 and then continually declined to near steady-state rates between 20-30 mcg/day. The percent release after 14 days was 2.9, 2.0 and 0.9 for ZFI 89, 52 and 122, respectively.

Conclusions: IVRs of 25mm diameter, determined to be the optimal size for macaques, were well tolerated and did not induce inflammation. Release of all ZFI compounds followed t 0.5 kinetics. These findings suggest that efficacy testing in primate models is warranted to fully evaluate the potential to prevent
transmission.

An exploration of the attitudes and strategic responses of soleproprietor micro-enterprises in adopting information and communication technology

This article analyses longitudinal case-based research exploring the attitudes and strategic responses of micro-enterprise owners in adopting information and communication technology (ICT). In so doing, it contributes to the limited literature on micro-enterprise ICT adoption, with a particular focus on sole proprietors. It provides a basis for widening the theoretical base of the literature pertaining to ICT adoption on two levels. First, a framework is developed which integrates the findings to illustrate the relationships between attitudes towards ICT adoption, endogenous and exogenous influencers of these attitudes and subsequent strategic response in ICT adoption. Second, building upon this framework the article reveals the unique challenges, opportunities and implications of ICT adoption for sole-proprietor micro-enterprises. © The Author(s) 2012

Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency, such that acetate (C2) has been described as FFA2 selective while propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations marked variation in ligand-independent, constitutive activity was identified. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity while the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the 2nd extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity, and in most cases also yielded corresponding changes in SCFA potency.

IHG-1 amplifies TGF-beta1 signaling and is increased in renal fibrosis

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.

Frequency and distribution of group I intron genotypes of Candida albicans colonising critically ill patients

A study is performed to examine the distribution and frequency of 25S rRNA intron genotypes of Candida albicans isolated from different anatomical sites of patients in an intensive care unit (ICU) setting. Germ-tube positive Candida isolates (n=65) from 65 patients are included and isolates are characterised by 25S intron genotyping, whereby all can be subdivided into four genotypes (A-D). Results demonstrated that there were no significant differences between the frequency and genotype distribution of the Candida isolates and the anatomical site of colonisation. Furthermore, analysis of the transposable intron region in the 25S rRNA gene demonstrated equal distribution, regardless of age and anatomical site of isolation (groin, throat, etc.). Therefore, there does not appear to be any selective pressure associated with any anatomical site, resulting in an ecological shift in the frequency of genotypes present. This suggests that C. albicans intron genotypes equally colonise those sites of the body examined in this study. Although such an ecological finding as this is interesting, it perpetuates the continued need to find a genotypic typing scheme that helps to identify the source (nosocomial or endogenous) and mode of entry of C. albicans into patients in the ICU setting, resulting in C. albicans bloodstream infection.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.

Deregulated estrogen metabolism in BRCA1 deficient cells induces chromosomal instability.

Objectives: Germline mutations in BRCA1 predispose carriers to a high
incidence of breast and ovarian cancers. The BRCA1 protein functions to maintain
genomic stability via important roles in DNA repair, transcriptional regulation, and
post-replicative repair. Despite functions in processes essential in all cells, BRCA1
loss or mutation leads to tumours predominantly in estrogen-regulated tissues.
Here, we aim to determine if endogenous estrogen metabolites may be an initiator
of genomic instability in BRCA1 deficient cells.

Methods: We analysed DNA DSBs by ?H2AX, 53BP1, and pATM1981
foci and neutral comet assay, estrogen metabolite concentrations by LC-MS/MS,
and BRCA1 transcriptional regulation of metabolism genes by ChIP-chip, ChIP,
and qRT-PCR.

Results: We show that estrogen metabolism is perturbed in BRCA1 deficient
cells resulting in elevated production of 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), and decreased production of the protective metabolite
4-methoxyestradiol. We demonstrate that 2-OHE2 and 4-OHE2 treatment leads
to DNA double strand breaks (DSBs) in breast cells, and these DSBs were exacerbated
in both BRCA1 depleted cells and BRCA1 heterozygous cells (harbouring
185delAG mutation). Furthermore, the DSBs were not repaired efficiently in either
BRCA1 depleted or heterozygous cells, and we found that 2-OHE2 and 4-OHE2
treatment generates chromosomal aberrations in BRCA1 depleted cells. We suggest
that the increase in DNA DSBs in BRCA1 deficient cells is due to loss of
both BRCA1 transcriptional repression of estrogen metabolising genes (such as
CYP1A1 and CYP3A4) and loss of transcriptional activation of detoxification
genes (such as COMT).

Conclusions: We suggest that BRCA1 loss results in estrogen driven tumourigenesis
through a combination of increased expression of estrogen metabolising
enzymes and reduced expression of protective enzymes, coupled with a defect in
the repair of DNA DSBs induced by endogenous estrogen metabolites. The overall
effect being an exacerbation of genomic instability in estrogen regulated tissues in
BRCA1 mutation carriers.

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 µg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.

Therapeutic potential of targeting lipid aldehydes and lipoxidation end-products in the treatment of ocular disease

Lipoxidation reactions and the subsequent accumulation of advanced lipoxidation end products (ALEs) have been implicated in the pathogenesis of many of the leading causes of visual impairment. Here, we begin by outlining some of the major lipid aldehydes produced through lipoxidation reactions, the ALEs formed upon their reaction with proteins, and the endogenous aldehyde metabolizing enzymes involved in protecting cells against lipoxidation mediated damage. Discussions are subsequently focused on the clinical and experimental evidence supporting the contribution of lipid aldehydes and ALEs in the development of ocular diseases. From these discussions, it is clear that inhibition of lipoxidation reactions and ALE formation could represent a new therapeutic avenue for the treatment of a broad range of ocular disorders. Current and emerging pharmacological strategies to prevent or neutralize the effects of lipid aldehydes and ALEs are therefore considered, with particular emphasis on the potential of these drugs for treatment of diseases of the eye.

The Anti-Migratory Effects of FKBPL and Its Peptide Derivative, AD-01: Regulation of CD44 and the Cytoskeletal Pathway

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity and and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Citrate synthase from the liver fluke Fasciola hepatica

Citrate synthase catalyses the first step of the Krebs' tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely a-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-β1-induced fibrotic responses

TGF-ß1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-ß1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localization of IHG-1 is pivotal in amplification of TGF-ß1 signaling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-ß1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (?mts-IHG-1) repressed TGF-ß1 fibrotic signaling in renal epithelial cells. In cells expressing ?mts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-ß1. ?mts-IHG-1 modulation of TGF-ß1 signaling was associated with increased Smad7 protein expression. ?mts-IHG-1 modulated TGF-ß1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-ß1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-ß1 signal transduction and that IHG-1 is a key player in this modulation.