Sequence-Based Hepatitis B Virus Antiviral Resistance Testing in Switzerland

BACKGROUND: A growing number of patients with chronic hepatitis B is being treated for extended periods with nucleoside and/or nucleotide analogs. In this context, antiviral resistance represents an increasingly common and complex issue. METHODS: Mutations in the hepatitis B virus (HBV) reverse transcriptase (rt) gene and viral genotypes were determined by direct sequencing of PCR products and alignment with reference sequences deposited in GenBank. RESULTS: Plasma samples from 60 patients with chronic hepatitis B were analyzed since March 2009. The predominant mutation pattern identified in patients with virological breakthrough was rtM204V/I ± different compensatory mutations, conferring resistance to L-nucleosides (lamivudine, telbivudine, emtricitabine) and predisposing to entecavir resistance (n = 18). Complex mutation patterns with a potential for multidrug resistance were identified in 2 patients. Selection of a fully entecavir resistant strain was observed in a patient exposed to lamivudine alone. Novel mutations were identified in 1 patient. Wild-type HBV was identified in 9 patients with suspected virological breakthrough, raising concerns about treatment adherence. No preexisting resistance mutations were identified in treatment-naïve patients (n = 13). Viral genome amplification and sequencing failed in 16 patients, of which only 2 had a documented HBV DNA > 1000 IU/ml. HBV genotypes were D in 28, A in 6, B in 4, C in 3 and E in 3 patients. Results will be updated in August 2010 and therapeutic implications discussed. CONCLUSIONS: With expanding treatment options and a growing number of patients exposed to nucleoside and/or nucleotide analogs, sequence-based HBV antiviral resistance testing is expected to become a cornerstone in the management of chronic hepatitis B.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters.

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional Löwenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.

Ku-deficient yeast strains exhibit alternative states of silencing competence.

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension.

Excessive proliferation of vascular wall cells underlies the development of elevated vascular resistance in hypoxic pulmonary hypertension (PH), but the responsible mechanisms remain unclear. Growth-promoting effects of catecholamines may contribute. Hypoxemia causes sympathoexcitation, and prolonged stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs) induces hypertrophy and hyperplasia of arterial smooth muscle cells and adventitial fibroblasts. Catecholamine trophic actions in arteries are enhanced when other conditions favoring growth or remodeling are present, e.g., injury or altered shear stress, in isolated pulmonary arteries from rats with hypoxic PH. The present study examined the hypothesis that catecholamines contribute to pulmonary vascular remodeling in vivo in hypoxic PH. Mice genetically deficient in norepinephrine and epinephrine production [dopamine beta-hydroxylase(-/-) (DBH(-/-))] or alpha(1)-ARs were examined for alterations in PH, cardiac hypertrophy, and vascular remodeling after 21 days exposure to normobaric 0.1 inspired oxygen fraction (Fi(O(2))). A decrease in the lumen area and an increase in the wall thickness of arteries were strongly inhibited in knockout mice (order of extent of inhibition: DBH(-/-) = alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-)). Distal muscularization of small arterioles was also reduced (DBH(-/-) > alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-) mice). Despite these reductions, increases in right ventricular pressure and hypertrophy were not attenuated in DBH(-/-) and alpha(1B)-AR(-/-) mice. However, hematocrit increased more in these mice, possibly as a consequence of impaired cardiovascular activation that occurs during reduction of Fi(O(2)). In contrast, in alpha(1D)-AR(-/-) mice, where hematocrit increased the same as in wild-type mice, right ventricular pressure was reduced. These data suggest that catecholamine stimulation of alpha(1B)- and alpha(1D)-ARs contributes significantly to vascular remodeling in hypoxic PH.

Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment.

Survival responses in stressed organs

Apoptosis or programmed cell death is a regulated form of cell suicide executed by cysteine proteases, or "caspases", to maintain proper tissue homeostasis in multicellular organisms. Dysregulation of apoptosis leads to pathological complications including cancer, autoimmunity, neurodegenerative, and heart diseases. Beside their known function as the key executioners of apoptotic cell death, caspases were reported to mediate non-apoptotic functions. In this report we study the survival signals conveyed through caspase-3-mediated cleavage of Ras GTPase-activating proteins (RasGAP). Ubiquitously expressed, RasGAP senses caspase activity and controls the cell death/survival switch. RasGAP is cleaved once at low caspase activity and the generated N-terminal fragment (fragment N) induces a survival response by activating Ras/PI3K/Akt pathway. However, high caspase activity associated with increased stress leads to fragment Ν cleavage into fragments that do not mediate any detectable survival signals. In this thesis project we studied the role of fragment Ν in protecting stressed organs as well as in maintenance of their functionality. In response to stress in different organs, we found that mice lacking caspase-3 or unable to cleave RasGAP (Knock-In mice), and therefore unable to generate fragment N, were deficient in Akt activation and experienced increased apoptosis compared to wild-type mice. Augmented tissue damage and organ dysfunction in those mice highlight the importance of fragment Ν in activating Akt-mediated prosurvival pathway and in protection of organs during episodes of stress. In parallel we investigated the role of fragment Ν in regulating the activation of transcription factor NF-kB, a master regulator of inflammation. Sustained NF-kB activation may be detrimental by directly causing apoptosis or leading to a persistent damaging inflammation response. We found that fragment Ν is a potent inhibitor of NF-kB by favoring its nuclear export. Therefore, fragment Ν regulates NF-kB activity and contributes to a controlled response as well as maintenance of homeostasis in stressed cells. Importantly, these findings introduce new insights of how activated caspase-3 acts as a stress intensity sensor that controls cell fate by either initiating a fragment N- dependent cell resistance program or a cell suicide response. This identifies the pivotal role of fragment Ν in protection against patho-physiological damage, and encourages the development of therapies which aim to increase cell resistance to vigorous treatment. - L'apoptose, ou mort cellulaire programmée, est une forme contrôlée de suicide cellulaire exécuté par des protéines appelées caspases, dans le but de maintenir l'homéostasie des tissus sains dans les organismes multicellulaires. Un mauvais contrôle de l'apoptose peut mener à des pathologies comme le cancer, la neurodégénération et les maladies cardiaques et auto-immunes. En dehors de leur rôle connu d'exécutrices de l'apoptose, les caspases ont aussi été identifiées dans d'autres contextes non-apoptotiques. Dans ce projet, nous avons étudié les signaux de survie émis par le résultat du clivage de RasGAP par la caspase-3. Exprimée de façon ubiquitaire, RasGAP est sensible à l'activité de caspase-3 et contrôle la décision de la cellule à entreprendre la mort ou la survie cellulaire. A un taux d'activité faible, la caspase-3 clive RasGAP, ce qui mène à la génération d'un fragment N-terminal, appelé Fragment N, qui induit des signaux de survie via l'activation de la cascade Ras/PI3K/Akt. Cependant, lorsque l'activité de la caspase-3 augmente, le fragment N est clivé, ce qui a pour effet d'éliminer ces signaux de survie. Dans ce travail, nous avons étudié le rôle du Fragment N dans la protection des organes en état de stress et dans le maintien de leur fonctionnalité. En réponse à certains stress, nous avons découvert que les organes de souris n'exprimant pas la caspase-3 ou alors incapables de cliver RasGAP (souris Kl), et de ce fait n'ayant pas la possibilité de générer le Fragment N, perdaient leur faculté d'activer la protéine Akt et démontraient un taux d'apoptose plus élevé que des organes de souris sauvages. Le fait que les organes et tissus de ces souris manifestaient de graves dommages et dysfonctions met en évidence l'importance du Fragment N dans l'activation des signaux de survie via la protéine Akt et dans la neutralisation de l'apoptose induite par la caspase-3. En parallèle, nous avons investigué le rôle du Fragment N dans la régulation de l'activation de NF-kB, un facteur de transcription clé dans l'inflammation. Une activation soutenue de NF-kB peut être délétère par activation directe de l'apoptose ou peut mener à une réponse inflammatoire persistante. Nous avons découvert que le Fragment N, en favorisant l'export de NF-kB depuis le noyau, était capable de l'inhiber très efficacement. Le Fragment N régule donc l'activité de NF-kB et contribue au maintien de l'homéostasie dans des cellules stressées. Ces découvertes aident, de façon importante, à la compréhension de comment l'activation de la caspase-3 agit comme senseur de stress et décide du sort de la cellule soit en initiant une protection par le biais du fragment N, ou en induisant un suicide cellulaire. Cette étude définit le Fragment Ν comme ayant un rôle de pivot dans la protection contre des dommages patho-physiologiques, et ouvre des perspectives de développement de thérapies qui cibleraient à augmenter la résistance à divers traitements.

Functional Characterization of a n NTPase Activity of the Hepatitis C Virus Nonstructural Protein 4B

Background: Nonstructural p rotein 4 B (NS4B) i s the m asterorganizer of hepatitis C virus (HCV) replication complexformation. It is a multispanning membrane protein that has beenreported to p ossess NTPase activity. This enzymatic functionhas been poorly studied so far and its role in the HCV life cycleis u nknown. T he present w ork-in-progress a ims at validatingand functionally c haracterizing this a ctivity a nd its r ole in t heviral life cycle.Methods: B ioinformatic analyses were performed to i dentifykey residues for site-directed mutagenesis, both in t he contextof s ubgenomic r eplicons a s well as recombinant v iruses.Mutants were investigated with respect to R NA replication andinfectious particle p roduction. In p arallel, expression andpurification of recombinant wild-type and mutant NS4B proteinsare being pursued to characterize enzymatic activity in vitro.Results: B ioinformatic a nalyses revealed t hat p redictedNTPase features are conserved only in H CV NS4B b ut n ot i nNS4B from other Flaviviridae f amily m embers. A laninesubstitutions were designed to target predicted key Walker A, Band C motifs. These substitutions affected RNA replication andinfectious virus production to v arying degrees. Optimization ofrecombinant protein production is i n progress both in b acterialas well as mammalian expression systems.Conclusions: These studies should yield new insights into thefunctions of this hitherto poorly characterized viral nonstructuralprotein and may reveal novel targets for antiviral intervention inthe future.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Dual role of Bmi1 loss in the preservation of photoreceptor layers in the Rd1 mouse

Purpose: In the Rd1 and Rd10 mouse models of retinitis pigmentosa, a mutation in the Pde6ß gene leads to the rapid loss of photoreceptors. As in several neurodegenerative diseases, Rd1 and Rd10 photoreceptors re-express cell cycle proteins prior to death. Bmi1 regulates cell cycle progression through inhibition of CDK inhibitors, and its deletion efficiently rescues the Rd1 retinal degeneration. The present study evaluates the effects of Bmi1 loss in photoreceptors and Müller glia, since in lower vertebrates, these cells respond to retinal injury through dedifferentiation and regeneration of retinal cells. Methods: Cell death and Müller cell activation were analyzed by immunostaining of wild-type, Rd1 and Rd1;Bmi1-/- eye sections during retinal degeneration, between P10 and P20. Lineage tracing experiments use the GFAP-Cre mouse (JAX) to target Müller cells. Results: In Rd1 retinal explants, inhibition of CDKs reduces the amount of dying cells. In vivo, Bmi1 deletion reduces CDK4 expression and cell death in the P15 Rd1;Bmi1-/- retina, although cGMP accumulation and TUNEL staining are detected at the onset of retinal degeneration (P12). This suggests that another process acts in parallel to overcome the initial loss of Rd1;Bmi1-/- photoreceptors. We demonstrate here that Bmi1 loss in the Rd1 retina enhances the activation of Müller glia by downregulation of p27Kip1, that these cells migrate toward the ONL, and that some cells express the retinal progenitor marker Pax6 at the inner part of the ONL. These events are also observed, but to a lesser extent, in Rd1 and Rd10 retinas. At P12, EdU incorporation shows proliferating cells with atypical elongated nuclei at the inner border of the Rd1;Bmi1-/- ONL. Lineage tracing targeting Müller cells is in process and will determine the implication of this cell population in the maintenance of the Rd1;Bmi1-/- ONL thickness and whether downregulation of Bmi1 in Rd10 Müller cells equally stimulates their activation. Conclusions: Our results show a dual role of Bmi1 deletion in the rescue of photoreceptors in the Rd1;Bmi1-/- retina. Indeed, the loss of Bmi1 reduces Rd1 retinal degeneration, and as well, enhances the Müller glia activation. In addition, the emergence of cells expressing a retinal progenitor marker in the ONL suggests Bmi1 as a blockade to the regeneration of retinal cells in mammals.

Wet chemical silver treatment of endotracheal tubes to produce antibacterial surfaces.

Mechanically ventilated patients in hospitals are subjected to an increased risk of acquiring nosocomial pneumonia that sometimes has a lethal outcome. One way to minimize the risk could be to make the surfaces on endotracheal tubes antibacterial. In this study, bacterial growth was inhibited or completely prevented by silver ions wet chemically and deposited onto the tube surface. Through the wet chemical treatment developed here, a surface precipitate was formed containing silver chloride and a silver stearate salt. The identity and morphology of the surface precipitate was studied using x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray powder diffraction. Leaching of silver ions into solution was examined, and bacterial growth on the treated surfaces was assayed using Pseudomonas aeruginosa wild type (PAO1) bacteria. Furthermore, the minimum inhibitory concentration of silver ions was determined in liquid- and solid-rich growth medium as 23 and 18 microM, respectively, for P. aeruginosa.

Strength and limitations of regulatory T Cells for immunotherapy in transplantation.

In many experimental models, CD4+CD25+Foxp3+ regulatory T cells (nTreg) have been identifi ed as key players in promoting peripheral transplantation (Tx) tolerance. We have been focusing on therapies based on antigen-specifi c nTreg that can control effector T cells (Teff) and prevent allograft rejection. The use of nTreg in immunotherapeutic protocols for solid organ Tx is however limited by their overall low numbers as well as the low precursor frequency of alloantigen cross-reactive nTreg expected to be found in a normal individual. Moreover, although we previously described robust protocols to generate and expand antigen-specifi c nTreg in vitro, the process requires careful selection of highly pure nTreg and cumbersome ex-vivo manipulations, rendering this strategy not easily applicable in clinical solid organ Tx. In this study, we aimed to expand Treg directly in vivo and determine their suppressive function, effi cacy and stability in promoting donor-specifi c tolerance in a stringent murine Tx model. Our data suggest that IL-2-based therapies lead to a signifi cant increase of Treg in vivo. The expanded Treg suppressed Teff proliferation (albeit slightly less effi ciently than nTreg isolated from control mice) and allowed prolonged graft survival of major MHC-mismatched skin grafts in wild-type non-lymphopenic recipients. The expanded Treg alone were however not suffi cient to induce tolerance in stringent experimental conditions. Rapamycin reduced the frequency of Teff but did not impede expansion of Treg. Pro-infl ammatory stimuli hindered the expansion of Treg and resulted in an increase in the frequency of CD4+IFN-γ+ and CD4+IL17+ T cells. We propose that IL-2-based treatments would be an effi cient method for expanding functional Treg in vivo without affecting other immune cell populations, thereby favorably shifting the pool of alloreactive T cells towards regulation in response to an allograft. However, we also highlight some potential limitations of Treg expansion such as concomitant infl ammatory events.