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Optical microelectrodes (optrodes) are used in neuroscience to transmit light into the brain of a genetically modified animal to evoke and record electrical activity from light-sensitive neurons. Our novel micro-optrode solution integrates a light-transmitting 125 micrometer optical fiber and a 9 micrometer carbon monofilament to form an electrical lead element, which is contained in a borosilicate glass sheathing coaxial arrangement ending with a micrometer-sized carbon tip. This novel unit design is stiff and slender enough to be used for targeting deep brain areas, and may cause less tissue damage compared with previous models. The center-positioned carbon fiber is less prone to light-induced artifacts than side-lit metal microelectrodes previously presented. The carbon tip is capable of not only recording electrical signals of neuronal origin but can also provide valuable surface area for electron transfer, which is essential in electrochemical (voltammetry, amperometry) or microbiosensor applications. We present details of design and manufacture as well as operational examples of the newly developed single micro-optrode, which includes assessments of 1) carbon tip length-impedance relationship, 2) light transmission capabilities, 3) photoelectric artifacts in carbon fibers, 4) responses to dopamine using fast-scan cyclic voltammetry in vivo, and 5) optogenetic stimulation and spike or local field potential recording from the rat brain transfected with channelrhodopsin-2. With this work, we demonstrate that our novel carbon tipped single micro-optrode may open up new avenues for use in optogenetic stimulation when needing to be combined with extracellular recording, electrochemical, or microbiosensor measurements performed on a millisecond basis.

The GIHU004 study was designed to evaluate the safety and immunogenicity of three doses of DermaVir immunization in HIV-infected subjects on fully suppressive combination antiretroviral therapy (cART).This first-in-human dose escalation study was conducted with three topical DermaVir doses targeted to epidermal Langerhans cells to express fifteen HIV antigens in draining lymph nodes: 0.1 mg DNA targeted to two, 0.4 mg and 0.8 mg DNA targeted to four lymph nodes. Particularly, in the medium dose cohort 0.1 mg DNA was targeted per draining lymph node via ∼8 million Langerhans cells located in 80 cm(2) epidermis area. The 28-days study with 48-week safety follow-up evaluated HIV-specific T cell responses against Gag p17, Gag p24 and Gag p15, Tat and Rev antigens. DermaVir-associated side effects were mild, transient and not dose-dependent. Boosting of HIV-specific effector CD4(+) and CD8(+) T cells expressing IFN-gamma and IL-2 was detected against several antigens in every subject of the medium dose cohort. The striking result was the dose-dependent expansion of HIV-specific precursor/memory T cells with high proliferation capacity. In low, medium and high dose cohorts this HIV-specific T cell population increased by 325-, 136,202 and 50,759 counts after 4 weeks, and by 3,899, 9,878 and 18,382 counts after one year, respectively, compared to baseline.Single immunization with the DermaVir candidate therapeutic vaccine was safe and immunogenic in HIV-infected individuals. Based on the potent induction of Gag, Tat and Rev-specific memory T cells, especially in the medium dose cohort, we speculate that DermaVir boost T cell responses specific to all the 15 HIV antigens expressed from the single DNA. For durable immune reactivity repeated DermaVir immunization might be required since the frequency of DermaVir-boosted HIV-specific memory T cells decreased during the 48-week follow up.ClinicalTrial.gov NCT00712530.

Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2+)-channels but not affecting motoneurons.

Purpose: The basis of the antitumor immunotherapy, of which the purpose is the stimulation of the immune system. We have used two of the Pathogen Associated Molecular Patterns: unmethylated CpG oligonucleotide, a ligand of Toll-like receptor 9 (TLR9), and lipopolysaccharide (LPS) which is recognized by TLR4, combined with an agonistic OX40 receptor-specific monoclonal antibody (anti-OX40), which is expressed by activated regulatory T-cells (and by other activated T-cell populations as well). The objective of this study was to prove the effectiveness of the aforementioned compounds in an animal model, on a bladder cancer cell line., Methods: We have instilled MB49 cells subcutaneously, to the left musculus biceps femoris. We have created three observation groups, each containing ten mice. After eleven days, all treated mice bearing the size of 5-8 mm (in diameter) tumor were administered CpG + anti-OX40 or LPS + anti-OX40 three times with a three-day lap between each treatment. Mice in the control group did not receive any treatment., Results: All the specimens from the control and LPS + anti-OX40 groups have died by the sixtieth day of the observation period, however, five mice from the CpG + anti-OX40 group were still alive. The experiment lasted until the last surviving mouse died, which occurred on the 357th day after tumor implantation., Discussion: The treatment with LPS did not make anti-OX40 more potent and did not increase the survival times. However, CpG + anti-OX40 has shown increased antitumor activity compared to the other two groups., Competing Interests: The authors declare that they have no conflict of interest regarding the publication of this manuscript.

The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94-99 and to amino acids 140-147 on the beta chain and it recognizes the amino acids 23-32 and 93-98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity., Competing Interests: We have the following interests. József Antal is employed by Diagon Ltd who declares that on the basis of the current result presented in this manuscript, a commercial product, namely Dia-D-Dimer IVD test has been developed. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.