Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

3-(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid: an allosteric inhibitor of fructose-1,6-bisphosphatase at the AMP site

3-(2-Carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid (MDL-29951), an antagonist of the glycine site of the NMDA receptor, has been found to be an allosteric inhibitor of the enzyme fructose 1,6-bisphosphatase. The compound binds at the AMP regulatory site by X-ray crystallography. This represents a new approach to inhibition of fructose 1,6-bisphosphatase and serves as a lead for further drug design.     

An efficient,widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.     

Placental Transfer of a Fully Human IgG2 Monoclonal Antibody in the Cynomolgus Monkey,Rat, and Rabbit: A Comparative Assessment from during Organogenesis to Late Gestation

Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo‐fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6‐fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3‐fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10‐fold early in the second trimester (gd50–70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6‐fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high‐dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans     

Single Administration of p2TA (AB103), a CD28 Antagonist Peptide,Prevents Inflammatory and Thrombotic Reactions and Protects against Gastrointestinal Injury in Total-Body Irradiated Mice

The goal of this study was to elucidate the action of the CD28 mimetic peptide p2TA (AB103) that attenuates an excessive inflammatory response in mitigating radiation-induced inflammatory injuries. BALB/c and A/J mice were divided into four groups: Control (C), Peptide (P; 5 mg/kg of p2TA peptide), Radiation (R; total body irradiation with 8 Gy γ-rays), and Radiation + Peptide (RP; irradiation followed by p2TA peptide 24 h later). Gastrointestinal tissue damage was evaluated by analysis of jejunum histopathology and immunohistochemistry for cell proliferation (Cyclin D1) and inflammation (COX-2) markers, as well as the presence of macrophages (F4/80). Pro-inflammatory cytokines IL-6 and KC as well as fibrinogen were quantified in plasma samples obtained from the same mice. Our results demonstrated that administration of p2TA peptide significantly reduced the irradiation-induced increase of IL-6 and fibrinogen in plasma 7 days after exposure. Seven days after total body irradiation with 8 Gy of gamma rays numbers of intestinal crypt cells were reduced and villi were shorter in irradiated animals compared to the controls. The p2TA peptide delivery 24 h after irradiation led to improved morphology of villi and crypts, increased Cyclin D1 expression, decreased COX-2 staining and decreased numbers of macrophages in small intestine of irradiated mice. Our study suggests that attenuation of CD28 signaling is a promising therapeutic approach for mitigation of radiation-induced tissue injury.     

Identification of Novel Genetic Risk Loci in Maltese Dogs with Necrotizing Meningoencephalitis and Evidence of a Shared Genetic Risk across Toy Dog Breeds

Necrotizing meningoencephalitis (NME) affects toy and small breed dogs causing progressive, often fatal, inflammation and necrosis in the brain. Genetic risk loci for NME previously were identified in pug dogs, particularly associated with the dog leukocyte antigen (DLA) class II complex on chromosome 12, but have not been investigated in other susceptible breeds. We sought to evaluate Maltese and Chihuahua dogs, in addition to pug dogs, to identify novel or shared genetic risk factors for NME development. Genome-wide association testing of single nucleotide polymorphisms (SNPs) in Maltese dogs with NME identified 2 regions of genome-wide significance on chromosomes 4 (chr4:74522353T>A, p = 8.1×10⁻⁷) and 15 (chr15:53338796A>G, p = 1.5×10⁻⁷). Haplotype analysis and fine-mapping suggests that ILR7 and FBXW7, respectively, both important for regulation of immune system function, could be the underlying associated genes. Further evaluation of these regions and the previously identified DLA II locus across all three breeds, revealed an enrichment of nominal significant SNPs associated with chromosome 15 in pug dogs and DLA II in Maltese and Chihuahua dogs. Meta-analysis confirmed effect sizes the same direction in all three breeds for both the chromosome 15 and DLA II loci (p = 8.6×10^–11 and p = 2.5×10⁻⁷, respectively). This suggests a shared genetic background exists between all breeds and confers susceptibility to NME, but effect sizes might be different among breeds. In conclusion, we identified the first genetic risk factors for NME development in the Maltese, chromosome 4 and chromosome 15, and provide evidence for a shared genetic risk between breeds associated with chromosome 15 and DLA II. Last, DLA II and IL7R both have been implicated in human inflammatory diseases of the central nervous system such as multiple sclerosis, suggesting that similar pharmacotherapeutic targets across species should be investigated.     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. METHODS: Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.     

Splenocytes from NZB mice exhibit spontaneously high rates of fusion compared to control strains

A previous report (G. E. Woloschak and D. Senitzer, submitted for publication) has demonstrated that mitogen-stimulated splenocytes fuse at a much higher frequency than untreated splenocytes as measured by the fusion index (a calculation of the number of nuclei in fused cells vs the total number of nuclei). A measurement of the fusion indices of NZB spleen cells provides results markedly different from those observed with splenocytes from control strains of mice—NZB spleen cells exhibit a spontaneously high fusion index. In this assay, they spontaneously display the same fusing capacity as that observed in cultures of mitogen-treated spleen cells from control strains of mice. This elevated fusion index is not affected by treatment with anti-Thy-1.2 and complement, but is abrogated by treatment with anti-immunoglobulin serum and complement. This suggests that a B cell is responsible for the high fusion index of NZB splenocytes. This high fusion index is present when using splenocytes from both male and female mice in the fusion assay, and can be observed using spleen cells from NZB mice as young as 12 days. This appears to be the result of a spontaneous polyclonal B-cell activation in NZB mice.