Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index ≥2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-γ (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.     

Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation

Background

With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.

Methods and Results

Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.

Conclusions

We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.     

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

From symmetrical to asymmetrical nitrido phosphino-thiol complexes: a new class of neutral mixed-ligand (99m)Tc compounds as potential brain imaging agents

A general procedure is presented for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core that could be used to design either essential or target specific imaging agents. This procedure is based on the chemical properties of a new monosubstituted [Tc(N)(R2PS)Cl(PPh3)] species composed of a TcN multiple bond and an ancillary phosphine thiol ligand (R2PSH). This intermediate readily reacts with bidentate mononegative ligands (S--Y) containing soft pi-donor coordinating atoms to give neutral pentacoordinate asymmetrical complexes of the type [Tc(N)(R2PS)(S--Y)]. The ability of several bidentate ligands containing different combination of heteroatoms (S, N, O) to form complexes with the [Tc(N)(R2PS)]+ building block was investigated. It was found that mononegative dithiocarbamate (DTC) or cysteine carboxyl derivate ligands promptly react with the monosubstituted species to form the final mixed compound in high yield. Preliminary biodistribution data in rats of some representative [Tc(N)(R2PS)(DTC)] compounds revealed an interesting initial brain uptake (in the range 0.20 +/- 0.01% ID/g and 0.91 +/- 0.06% ID/g), indicating their ability to cross in and out of the intact BBB. In these complexes the dithiocarbamate, or more generally the bidentate ligand (S--Y), can be designed to carry a functional group or a bioactive molecule, which could be involved in a trapping mechanism to increase brain retention for longer time intervals. These results could be conveniently utilized to devise a new procedure for the production of a novel class of brain perfusion and/or brain receptor imaging agents.     

Role of Hematopoietic Cells in the Maintenance of Chronic Human Torquetenovirus Plasma Viremia

Many aspects of the life cycle of torquetenoviruses (TTVs) are essentially unexplored. In particular, it is still a matter of speculation which cell type(s) replicates the viruses and maintains the generally high viral loads found in the blood of infected hosts. In this study, we sequentially measured the TTV loads in the plasma of four TTV-positive leukemia patients who were strongly myelosuppressed and then transplanted with haploidentical hematopoietic stem cells. The findings provide clear quantitative evidence for an extremely important role of hematopoietic cells in the maintenance of TTV viremia.Torquetenoviruses (TTVs) are small naked DNA viruses distinguished by a circular single-stranded DNA genome of only 3.8 kb, classified within the newly established family Anelloviridae (7). TTVs have been found in several animal species but do not appear capable of interspecies transmission. Due to their extensive genetic heterogeneity, human TTVs have been operatively subdivided into 5 genogroups and more than 40 genotypes (4). A remarkable feature of these TTVs is their presence in the plasma of nearly all people, regardless of geographical origin, age, and health status, raising many questions about their life cycle and possible pathological implications (2, 5). Plasma loads of TTVs vary extensively in both healthy and diseased individuals, usually ranging between 10³ and 10⁷ DNA copies per ml of plasma. However, some patients, including those with selected inflammatory or neoplastic disorders, transplant recipients, and human immunodeficiency virus-infected individuals, have a tendency to carry especially high burdens of TTVs (1, 6, 13, 22-24).By studying the dynamics of TTV viremia in individuals treated with alpha interferon for hepatitis C, the kinetics of virus replication was found to be quite high, with numbers of virions released into plasma and cleared from it daily on the same order of magnitude as other chronic plasma viremia-inducing viruses, such as the hepatitis B, hepatitis C, and human immunodeficiency viruses (16). Yet, due to considerable difficulties encountered in propagating TTVs in culture and in distinguishing the virions passively adsorbed onto the cells from the ones replicating inside cells, the tissue or tissues where these large numbers of TTV virions originate have yet to be established. Given that the amino acid compositions of the capsid protein believed to mediate viral adsorption to cells are quite diverse in different TTVs (2, 3, 9), it is also possible that permissive cells vary depending on the TTV considered. Relevant studies are limited. Short-term cultures of phytohemagglutinin-stimulated peripheral lymphocytes, but not resting lymphocytes were found to permit a measurable level of TTV replication (15, 18), indicative of at least a moderate degree of lymphotropism. On the other hand, the detection of replicative forms of TTV DNA in several tissues, including bone marrow, peripheral blood mononuclear cells, and liver, has suggested that TTVs might be polytropic in nature (2, 21).In 1999, Kanda et al. (10), researching TTV plasma of bone marrow transplant recipients with a qualitative PCR, noticed that 5 out of 6 previously positive patients tested negative in a sampling collected during the myelosuppressed period and became positive again after graft reconstitution, leading them to suggest that TTV might replicate mainly in hematopoietic cells. In the present study, we further developed this observation by measuring the TTV load in sequential plasma samples obtained from four TTV-positive leukemia patients undergoing hematopoietic stem cell transplantation. This procedure basically consists of a myeloablative conditioning regimen (chemotherapy plus radiotherapy) followed by reinfusion of a positively selected CD34⁺ stem cell population. The findings are of interest because, in addition to confirming the decrease of TTV load observed by Kanda et al., they shed light on the kinetics of the effect, thus providing a better insight onto the role of hematopoietic cells in the maintenance of TTV viremia and on the life cycle of TTV in general.Table ​Table11 summarizes the main characteristics of the patients selected for the study. They were treated with 10 Gy total-body irradiation (TBI) on day 0 and received 5 mg/kg/day thiotepa on days 2 and 3, 40 mg/m²/day fludarabine on days 3 to 7, and 1.2 mg/kg/day antithymocyte globulin on days 4 to 8, and then, on day 10, they received the indicated numbers of positively selected CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. Peripheral blood samples were collected for TTV studies immediately before TBI and at selected times for the next 30 days, and plasma was stored in aliquots at −80°C until DNA extraction. The assay used for TTV quantification was a previously described highly sensitive TaqMan real-time PCR having the potential to detect and quantitate all hitherto recognized genetic forms of the virus (15, 16). All samples from each patient were assayed in a single run and in triplicate, and at least two independent DNA extractions for each sample were examined. The DNA extracts obtained at time zero were also typed with a previously described panel of five distinct PCR assays (12), each specific for one of the genogroups into which TTVs are subdivided. At the start of the study, the patients had viral loads ranging from 4.7 to 6.8 log copies per ml of plasma and harbored between 1 and 3 TTV genogroups (Table ​(Table1).1). In particular, all carried genogroup 1, which is highly represented in our area (12), and two carried one or two further genogroups. Consistent with previous findings (12), the patient who harbored three genogroups was the one with the highest viral load. As shown by Fig. ​Fig.1,1, in all four patients, TBI was followed by a steady decline of TTV viremia that continued for at least 22 days and progressively brought the virus to levels very close to the detection limit of the detection/quantitation method used, corresponding to values ranging between 0.003 (patient 3) and 0.00009 (patient 1) of the loads present prior to TBI. However, in no instance did the viral loads go below the limit of sensitivity of the assay (2 × 10² TTV DNA copies per ml of plasma). Although the size of the study does not permit firm conclusions on this aspect, it is noteworthy that the extent of decline was unrelated to the type and number of infecting TTV genogroup(s) originally present in the patients.Open in a separate windowFIG. 1.Plasma TTV loads and WBC counts in the peripheral blood of the 4 patients (Pt. 1 to 4) enrolled in the study. The arrow indicates the day the patients were infused with CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. The horizontal broken line represents the lower limit of sensitivity of the TTV detection method used.

TABLE 1.

Relevant parameters of the patients enrolled

Rac3-induced neuritogenesis requires binding to Neurabin I

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.     

Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Alcohol Affects the Brain's Resting-State Network in Social Drinkers

Acute alcohol intake is known to enhance inhibition through facilitation of GABA_A receptors, which are present in 40% of the synapses all over the brain. Evidence suggests that enhanced GABAergic transmission leads to increased large-scale brain connectivity. Our hypothesis is that acute alcohol intake would increase the functional connectivity of the human brain resting-state network (RSN). To test our hypothesis, electroencephalographic (EEG) measurements were recorded from healthy social drinkers at rest, during eyes-open and eyes-closed sessions, after administering to them an alcoholic beverage or placebo respectively. Salivary alcohol and cortisol served to measure the inebriation and stress levels. By calculating Magnitude Square Coherence (MSC) on standardized Low Resolution Electromagnetic Tomography (sLORETA) solutions, we formed cortical networks over several frequency bands, which were then analyzed in the context of functional connectivity and graph theory. MSC was increased (p<0.05, corrected with False Discovery Rate, FDR corrected) in alpha, beta (eyes-open) and theta bands (eyes-closed) following acute alcohol intake. Graph parameters were accordingly altered in these bands quantifying the effect of alcohol on the structure of brain networks; global efficiency and density were higher and path length was lower during alcohol (vs. placebo, p<0.05). Salivary alcohol concentration was positively correlated with the density of the network in beta band. The degree of specific nodes was elevated following alcohol (vs. placebo). Our findings support the hypothesis that short-term inebriation considerably increases large-scale connectivity in the RSN. The increased baseline functional connectivity can -at least partially- be attributed to the alcohol-induced disruption of the delicate balance between inhibitory and excitatory neurotransmission in favor of inhibitory influences. Thus, it is suggested that short-term inebriation is associated, as expected, to increased GABA transmission and functional connectivity, while long-term alcohol consumption may be linked to exactly the opposite effect.