Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

Background: Measurement of concentrations of acute-phase proteins (APPs) is used as an aid in the diagnosis of a variety of diseases in animals.Objective: To determine the concentration of APPs in dogs with steroid responsive meningitis-arteritis (SRMA) and other neurologic diseases.Animals: One hundred and thirty-three dogs with neurologic diseases, 6 dogs with sepsis, and 8 healthy dogs were included in the study. Thirty-six dogs had SRMA (31 of which had monitoring), 14 dogs had other meningoencephalitides (ME), 32 had disk disease (IVDD/DLSS), 26 had tumors affecting the central nervous system (TCNS), and 25 had idiopathic epilepsy (IE).Methods: Prospective, observational study: C-reactive protein (CRP), a 2 -macroglobulin (AMG), and albumin concentrations were determined in the serum or plasma. CRP was also measured in the cerebrospinal fluid.Results: Serum CRP was significantly higher in dogs with SRMA (x 5 142 mg/mL AE 75) and sepsis (x 5 114 mg/mL AE 67) in comparison with dogs with other neurologic diseases (x 5 2.3-21 mg/mL; P o .001). There was no significant difference detected in AMG between groups. Serum albumin concentration was significantly lower (P o .01) in dogs with SRMA (x 5 3.2 g/dL AE 0.41) than in other groups (x 5 3.6-3.9 g/dL). Serum CRP concentration of SRMA dogs correlated with alkaline phosphatase levels (r 5 0.515, P 5 .003).Conclusions and Clinical Importance: CRP concentrations in serum are useful in diagnosis of dogs with SRMA. Serum CRP could be used as a monitoring parameter in treatment management of these dogs.

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Spinal cord injury (SCI) in dogs is a well recognized animal model to study pathogenesis and treatment modalities of the debilitating human disease. To define the contributing role of microglial cell activation to the secondary wave following SCI, microglia from 15 dogs with SCI confirmed by imaging, gross, and histopathological examination were isolated and characterized in terms of morphology, immunophenotype, and function ex vivo by flow cytometry, allowing single cell analysis. The results were compared to region-specific findings obtained from healthy control dogs. Light microscopy revealed a significant enhancement of myelinophagia within the traumatized spinal cord of dogs who had had SCI for ≥5 days. Immunophenotypical characterization revealed increased expression of B7-1, B7-2, MHC II, CD1c, ICAM 1, CD14, CD44, and CD45 emphasizing the enhanced function of microglia as co-stimulators of T cells, in leukocyte adhesion and aggregation, and for lipid or glycolipid presentation. In addition, phagocytosis and reactive oxygen species (ROS) generation were significantly increased in dogs with spinal cord trauma. Regional differences within the spinal cord were observed by demonstrating disparities in microglial immunophenotypes in the traumatized cervical compared to the thoracolumbar spinal cord. In contrast to histopathology, microglia activation analyzed on a single cell basis did not depend upon the time span following SCI.

BackgroundSteroid-responsive meningitis-arteritis (SRMA) is an immune-mediated disorder characterized by neutrophilic pleocytosis and an arteritis particularly in the cervical leptomeninges. Previous studies of the disease have shown increased levels of IL-6 and TGF-ß1 in cerebrospinal fluid (CSF). In the presence of these cytokines, naive CD4+ cells differentiate into Th17 lymphocytes which synthesize interleukin 17 (IL-17). It has been shown that IL-17 plays an active role in autoimmune diseases, it induces and mediates inflammatory responses and has an important role in recruitment of neutrophils. The hypothesis of a Th17-skewed immune response in SRMA should be supported by evaluating IL-17 and CD40L, inducing the vasculitis.MethodsAn enzyme-linked immunosorbent assay (ELISA) was performed to measure IL-17 and CD40L in serum and CSF from a total of 79 dogs. Measurements of patients suffering from SRMA in the acute state (SRMA A) were compared with levels of patients under treatment with steroids (SRMA T), recurrence of the disease (SRMA R), other neurological disorders, and healthy dogs, using the two-part test. Additionally, secretion of IL-17 and interferon gamma (IFN-γ) from the peripheral blood mononuclear cells (PBMCs) was confirmed by an enzyme-linked immunospot (ELISpot) assay.ResultsSignificant higher levels of IL-17 were found in CSF of dogs with SRMA A compared with SRMA T, other neurological disorders and healthy dogs (p < 0.0001). In addition, levels of CD40L in CSF in dogs with SRMA A and SRMA R were significantly higher than in those with SRMA T (p = 0.0004) and healthy controls (p = 0.014). Furthermore, CSF concentrations of IL-17 and CD40L showed a strong positive correlation among each other (rSpear = 0.6601; p < 0.0001) and with the degree of pleocytosis (rSpear = 0.8842; p < 0.0001 and rSpear = 0.6649; p < 0.0001, respectively). IL-17 synthesis from PBMCs in SRMA patients was confirmed; however, IL-17 is mainly intrathecally produced.ConclusionsThese results imply that Th17 cells are inducing the autoimmune response in SRMA and are involved in the severe neutrophilic pleocytosis and disruption of the blood-brain barrier (BBB). CD-40L intrathecal synthesis might be involved in the striking vasculitis. The investigation of the role of IL-17 in SRMA might elucidate important pathomechanism and open new therapeutic strategies.