Rod and cone pathway signalling is altered in the P2X7 receptor knock out mouse.

The P2X7 receptor (P2X7-R) is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO) mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.

The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Activity-dependent expression of RNA binding protein HuD and its association with mRNAs in neurons.

The dendritic trafficking of RNA binding proteins (RBPs) is an important posttranscriptional process involved in the regulation of synaptic plasticity. For example, HuD RBP binds to AU-rich elements (AREs) in the 3' untranslated regions (3'UTR) of immediate-early gene (IEG) transcripts, whose protein products directly affect synaptic plasticity. However, the subcellular localization of HuD RBPs and associated mRNAs has not been investigated following neuronal stimulation. Immunofluorescence analysis revealed activity-dependent dendritic localization of HuD RBPs following KCl stimulation in hippocampal neurons, while immunoprecipitation demonstrated the association of HuD RBP with neuronal mRNAs encoding neuritin, Homer1a, GAP-43, Neuroligins, Verge and CAMKIIalpha. Activity-dependent expression of HuD involves activation of NMDAR as NMDA receptor 1 knockout mice (Nr1(neo-/-)) exhibited decreased expression of HuD. Moreover, translational regulation of HuD-associated transcripts was suggested by its co-localization with poly-A-binding protein (PABP) as well as the cap-binding protein (eIF4E). We propose that post-transcriptional regulation of neuronal mRNAs by HuD RBPs mediates protein synthesis-dependent changes in synaptic plasticity.

Concomitant quantification of targeted drug delivery and biological response in individual cells.

Targeted therapies result in heterogeneous drug delivery, often with highly variable drug uptake in the targeted cells and significant numbers of cells that are essentially untargeted. However both the variably targeted cells and neighboring bystander cells may respond to the treatment. Using ionizing radiation as an example of a targeted therapeutic agent, we describe a quantitative immunofluorescence-based approach for concomitant quantification of exposure and measurement of biological responses in both targeted and bystander cells. Cultures of human skin fibroblasts are co-pulse-labeled with 3H-deoxycytidine (3H-dC) and bromodeoxyuridine (BrdU). The labeled cells, identified by BrdU immunofluorescence, are internally irradiated by low-energy beta-particles emitted by incorporated 3H-dC. BrdU immunofluorescence intensity is proportional to radioactivity incorporated and, therefore, to radiation dose rate. Cell-cycle arrest in G2 is measured in labeled cells as function of dose rate. Stress responses in bystander cells, indicated by a G1 checkpoint, are concomitantly measured with a flow cytometric-cumulative labeling index (FCM-CLI) assay. The overall approach presented herein may be useful in the context of evaluating responses to targeted drug delivery.

Modified and simplified western blotting protocol: use of intermittent microwave irradiation (IMWI) and 5% skim milk to improve binding specificity.

A modified western blotting protocol was developed to increase the binding specificity of antigens and antibodies, using intermittent microwave irradiation (IMWI) with seven antibodies and two cell lines. The method was based on IMWI of the blotting membrane in the immunoblotting step using 5% skim milk as the diluting buffer. For some antibodies against p53, CDK4 and cyclinE, there were no distinct differences between the IMWI(+) and IMWI(-) counterparts; but improvement over the standard protocol was noted in both. For some antibodies, such as the polyclonal antibody against tubulin and the monoclonal antibodies against beta-tubulin, cyclinA and cyclinB1 (which were otherwise difficult to obtain good results with), IMWI was extremely effective, resulting in clear, specifically binding bands and a clean background. Moreover, the times were reduced from 8 to 3 h. Both the IMWI(+) and IMWI(-) protocols can be applied as simple, rapid and highly specific detection techniques for applications with various antigens, reducing background 'noise' to a minimum.

131I-Lym-1 in mice implanted with human Burkitt's lymphoma (Raji) tumors: loss of tumor specificity due to radiolysis.

UNLABELLED: Preliminary evaluations of 125I-labeled Lym-1, an anti-lymphoma mouse IgG2a monoclonal antibody, demonstrated favorable tumor uptake in mice bearing human Burkitt's lymphoma (Raji) tumors. In this study, the pharmacokinetics of 125I- and 131I-Lym-1, and the dosimetry, efficacy, and toxicity of 131I-Lym-1 in Raji-tumored mice were evaluated. METHODS: Lym-1 was radioiodinated by the chloramine-T method and analyzed for monomeric fraction and immunoreactivity (antigen cell binding, relative to unmodified Lym-1). Nude mice bearing Raji tumors (20-500 mm3) received 1.5 MBq (40 microCi) 125I-Lym-1, or 1.5, 7.4, 14.8, or 18.5 MBq (40, 200, 400, or 500 microCi) 131I-Lym-1. Pharmacokinetic data (total body and blood clearance and biodistribution) were used to estimate radiation dosimetry. Mini-thermoluminescent dosimetry (TLD) was also used to measure radiation dosimetry directly for 7 days after injection of 131I-Lym-1. Tumor size, survival, body weight, and blood counts were monitored for 60 days to evaluate therapeutic efficacy and toxicity of 131I-Lym-1. RESULTS: At the time of injection, the mean quality assurance (QA) values for 125I-Lym-1 were 100% monomer and 100% relative immunoreactivity; the corresponding values for 131I-Lym-1 were 73% and 66%, indicating that radiolysis had occurred during the interval between radiolabeling and injection. 125I-Lym-1 exhibited high and sustained concentration in tumors relative to normal organs, whereas 131I-Lym-1 did not. Assuming identical pharmacokinetic behavior to 125I-Lym-1, 131I-Lym-1 would deliver radiation doses of 3.45, 0.83, 1.03, 0.34, and 0.56 Gy per MBq injected (12.8, 3.1, 3.8, 1.3, and 2.1 rad/microCi), to tumor, liver, lungs, total body, and marrow, respectively. When the actual pharmacokinetic data for 131I-Lym-1 (1.5 MBq) were used to estimate dosimetry, corresponding values of 0.51, 0.72, 0.49, 0.31, and 0.41 Gy/MBq (1.9, 2.7, 1.8, 1.1, and 1.5 rad/microCi) were obtained. Similar values were obtained for mice receiving 7.4 or 14.8 MBq of 131I-Lym-1. Similarly, TLD data indicated little preferential radiation dosimetry to tumor. Response rates (cure + CR + PR) for mice receiving 0, 7.4, 14.8, and 18.5 MBq of 131I-Lym-1 were 8%, 7%, 21%, and 45%, respectively. The LD50/30 dose of 131I-Lym-1 was 12.7 MBq (343 microCi). CONCLUSIONS: 125I-Lym-1 exhibited high and sustained concentration in Raji tumors in mice, indicating excellent therapeutic potential for 131I-Lym-1. However, in vitro QA results for 131I-Lym-1 indicated that radiolysis had occurred, and 131I-Lym-1 demonstrated little accumulation in tumor, or preferential radiation dosimetry to tumor in the same model.

[Specific antimicrobial immunity in patients with suppurative soft-tissue diseases undergoing conventional and modified ultraviolet irradiation of autologous blood]. / Spetsificheskii antimikrobnyi immunitet u bol'nykh s gnoinymi zabolevaniiami miagkikh tkanei pri obychnom i modifitsirovannom ul'trafioletovom obluchenii autokrovi.

Study of titers of specific antibacterial antibodies in the plasma by passive hemagglutination test and of the number of antibody-producing cells (APC) by local hemolysis test in patients with purulent diseases of soft tissues showed that both values increased after UV exposure of autoblood. This increase was more expressed after differentiated exposure of erythrocytic and leukocytic mass than after common UV exposure, which is explained by more complete exposure of leukocytes participating in immunological reactions. Study of APC during a UV session showed an increase in the count of these cells 6-8 h after exposure.

Activity of antibodies against Salmonella dublin, Toxoplasma gondii, or Actinobacillus pleuropneumoniae in sera after treatment with electron beam irradiation or binary ethylenimine.

Viral contamination of biological material may constitute a risk when samples are exchanged between countries, and it may be necessary to subject the material to an inactivation treatment. The present study investigated possible adverse effects on antibody activity subsequent to either electron beam irradiation or binary ethylenimine (BEI) treatment. The treatments were performed with sera obtained from pigs or cattle. For each treatment level, the posttreatment activity was plotted against the pretreatment activity, and regression analyses were carried out. The slope of the regression line was used as an estimate for the relative posttreatment activity. For a Toxoplasma gondii indirect enzyme-linked immunosorbent assay (ELISA) and agglutination assay as well as for a Salmonella dublin indirect ELISA, the posttreatment activity was more than 89% of the pretreatment activity when the samples were irradiated in the frozen state (on dry ice) with up to 46. kGy or when they were treated with 5 or 10 mM BEI for up to 48 h. The samples were more sensitive to irradiation in the liquid state. Thus, samples irradiated with 22.6 kGy retained 98% of their activity in the indirect ELISA when they were irradiated in the frozen state on dry ice but only 35% of their activity when they were irradiated in the liquid state at 0 degrees C. The patterns seen in an S. dublin blocking ELISA and an Actinobacillus pleuropneumoniae complement fixation assay differed in that samples with a low level of pretreatment activity were subject to a relatively greater decrease in activity than samples with a high level of pretreatment activity. The complement fixation assay was particularly sensitive to irradiation of serum. It is concluded that serum samples retain sufficient activity by both methods of virus inactivation, especially when used in indirect ELISA or in the T. gondii agglutination assay.