Validation of sensitivity to praziquantel using Schistosoma mansoni worm muscle tension and Ca2+-uptake as possible in vitro correlates to in vivo ED50 determination

Schistosome worm muscle tension and [45Ca2+]-uptake were tested as possible correlates of susceptibility to praziquantel (PZQ) assessed by estimating the drug ED50. Schistosoma mansoni cercariae of PZQ sensitive (S-CD, S-MOC and S-GP) and insensitive S. mansoni isolates (I-EE2, I-BANL and I-Senegal 47) were used to infect batches of CD-1 Swiss albino mice. Seven weeks after infection, animals of each batch were divided into six groups. Five of them received PZQ in doses of 12.5, 25, 50, 100 or 200 mg/kg PZQ, respectively, for five consecutive days, while the sixth was left as untreated controls. Two weeks after treatment mice were sacrificed, perfused and PZQ ED50's were estimated. Male worms recovered from infected untreated controls were examined for their muscle tension increase in response to PZQ using a physiological recorder coupled to a photooptic transducer. [45Ca2+]-uptake of male worms in the presence and absence of PZQ was determined using a liquid scintillation beta counter. Data revealed that PZQ insensitive isolates had significantly higher drug ED50 (>130 mg/kg) than PZQ sensitive isolates with ED50's <100 mg/kg. Moreover, in response to PZQ they were found to possess significant reductions in their worm muscle tension and their [45Ca2+]-uptake were <100%. Both parameters showed a significant negative correlation to PZQ ED50 in vivo and a significant positive correlation to each other.     

Innate immunity in multiple sclerosis: myeloid dendritic cells in secondary progressive multiple sclerosis are activated and drive a proinflammatory immune response

Multiple sclerosis (MS) is postulated to be a T cell-mediated autoimmune disease characterized clinically by a relapsing-remitting (RR) stage followed by a secondary progressive (SP) phase. The progressive phase is felt to be secondary to neuronal degenerative changes triggered by inflammation. The status of the innate immune system and its relationship to the stages of MS is not well understood. Dendritic cells (DCs) are professional APCs that are central cells of the innate immune system and have the unique capacity to induce primary immune responses. We investigated circulating myeloid DCs isolated directly from the blood to determine whether there were abnormalities in myeloid DCs in MS and whether they were related to disease stage. We found that SP-MS subjects had an increased percentage of DCs expressing CD80, a decreased percentage expressing PD-L1, and an increased percentage producing IL-12 and TNF-alpha compared with RR-MS or controls. A higher percentage of DCs from both RR and SP-MS patients expressed CD40 compared with controls. We then investigated the polarization effect of DCs from MS patients on naive T cells taken from cord blood using a MLR assay. Whereas DCs from RR-MS induced higher levels of Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4, IL-13) cytokines compared with controls, DCs from SP-MS only induced a polarized Th1 response. These results demonstrate abnormalities of DCs in MS and may explain the immunologic basis for the different stages and clinical patterns of MS.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

Molecular cloning and characterization of patellin1, a novel sec14-related protein, from zucchini (Cucurbita pepo)

A full-length patellin1 (PATL1) cDNA was cloned and characterized from zucchini (Cucurbita pepo). PATL1, originally discovered in the higher plant Arabidopsis thaliana, is a plant Sec14-related protein that localizes to the cell plate during the late stages of cytokinesis. PATL1 is related in sequence to other eukaryotic proteins involved in membrane trafficking and is thought to participate in vesicle trafficking events associated with cell plate maturation. The zucchini PATL1 (CpPATL1) cDNA predicts a 605 amino acid protein which consists of an acidic N-terminal domain (pI=4.2) followed by a Sec14 lipid-binding domain and a C-terminal Golgi dynamics domain (GOLD). The predicted CpPATL1 protein sequence shows a high degree of similarity to Arabidopsis PATL1, especially in the Sec14 (84%) and GOLD domains (87%). A phylogenetic analysis of all available full-length PATL sequences revealed that the PATLs belong to four distinct clades; CpPATL1 is a member of the PATL1/2 clade. RT-PCR analysis showed that the CpPATL1 gene is highly expressed throughout the plant. The domain structure, as well as biochemical fractionation studies, which demonstrated that CpPATL1 is a peripheral membrane protein, support a role in membrane trafficking events.     

Analysis of allelic variants in the catalase gene in patients with the skin depigmenting disorder vitiligo

Vitiligo is an acquired hypomelanotic skin disorder characterised by circumscribed depigmented macules resulting from the loss of functional melanocytes from the cutaneous epidermis. Conditions that might result in epidermal oxidative stress and consequently damage to pigment cells have been reported in the skin of vitiligo patients, including low catalase activity and increases in hydrogen peroxide levels. However, the cause of the decrease in catalase activity has not been equivocally determined. Several allelic variants in the catalase gene, a number of which have deleterious effects upon the expression or function of the enzyme, have been described and the aim of the present work was to assess the relevance of catalase gene variants in patients with vitiligo. Associations between ten separate allelic variants in the catalase gene and a predisposition to vitiligo were investigated in case-control studies with 166 English patients and 169 ethnically-matched controls using DNA sequencing and restriction fragment length polymorphism-polymerase chain reaction methods. Of the ten allelic variants analysed, only a C/T single nucleotide polymorphism in exon 9 of the catalase gene was associated with vitiligo. The C/T genotype was significantly over-represented in the vitiligo patient group compared with the control cohort. Of 166 vitiligo genotypes, 66 (39.8%) had the C/T variant compared to 45/169 (26.6%) control genotypes (P = 0.030). No evidence for an association between other allelic variants in the catalase gene and vitiligo susceptibility was found. The low catalase activity in vitiligo patient epidermis is more likely to result from environmental conditions such as inhibitory levels of hydrogen peroxide rather than allelic variations in the catalase gene which affect either expression or function of the enzyme.     

Auditory stimulus discrimination recorded in dogs, as indicated by mismatch negativity (MMN)

Dog cognition research tends to rely on behavioural response, which can be confounded by obedience or motivation, as the primary means of indexing dog cognitive abilities. A physiological method of measuring dog cognitive processing would be instructive and could complement behavioural response. Electroencephalogram (EEG) has been used in humans to study stimulus processing, which results in waveforms called event-related potentials (ERPs). One ERP component, mismatch negativity (MMN), is a negative deflection approximately 160-200 ms after stimulus onset, which may be related to change detection from echoic sensory memory. We adapted a minimally invasive technique to record MMN in dogs. Dogs were exposed to an auditory oddball paradigm in which deviant tones (10% probability) were pseudo-randomly interspersed throughout an 8 min sequence of standard tones (90% probability). A significant difference in MMN ERP amplitude was observed after the deviant tone in comparison to the standard tone, t₅ = −2.98, p = 0.03. This difference, attributed to discrimination of an unexpected stimulus in a series of expected stimuli, was not observed when both tones occurred 50% of the time, t₁ = −0.82, p > 0.05. Dogs showed no evidence of pain or distress at any point. We believe this is the first illustration of MMN in a group of dogs and anticipate that this technique may provide valuable insights in cognitive tasks such as object discrimination.     

Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.     

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.