Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei.

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA nT-nG , which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA nG , which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red-green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA nT-nG was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA nT-nG will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions.

A Self-Adjuvanted, Modular, Antigenic VLP for Rapid Response to Influenza Virus Variability.

The continuous evolution of influenza A virus (IAV) requires the influenza vaccine formulations to be updated annually to provide adequate protection. Recombinant protein-based vaccines provide safer, faster, and a more scalable alternative to the conventional embryonated egg approach for developing vaccines. However, these vaccines are typically poorer in immunogenicity than the vaccines containing inactivated or attenuated influenza viruses and require administration of a large antigen dosage together with potent adjuvants. The presentation of protein antigens on the surface of virus-like particles (VLP) provides an attractive strategy to rapidly induce stronger antigen-specific immune responses. Here we have examined the immunogenic potential and protective efficacy of P22 VLPs conjugated with multiple copies of the globular head domain of the hemagglutinin (HA) protein from the PR8 strain of IAV in a murine model of influenza pathogenesis. Using a covalent attachment strategy (SpyTag/SpyCatcher), we conjugated the HA globular head, which was recombinantly expressed in a genetically modified E. coli strain and found to refold as a monomer, to preassembled P22 VLPs. Immunization of mice with this P22-HAhead conjugate provided full protection from morbidity and mortality following infection with a homologous IAV strain. Moreover, the P22-HAhead conjugate also elicited an accelerated and enhanced HA head specific IgG response, which was significantly higher than the soluble HA head, or the admixture of P22 and HA head without the need for adjuvants. Thus, our results show that the HA head can be easily prepared by in vitro refolding in a modified E. coli strain, maintaining its intact structure and enabling the induction of a strong immune response when conjugated to P22 VLPs, even when presented as a monomer. These results also demonstrate that the P22 VLPs can be rapidly modified in a modular fashion, resulting in an effective vaccine construct that can generate protective immunity without the need for additional adjuvants.

A Novel Role for PDZ-Binding Motif of Influenza A Virus Nonstructural Protein 1 in Regulation of Host Susceptibility to Postinfluenza Bacterial Superinfections.

Influenza A viruses (IAVs) have multiple mechanisms for altering the host immune response to aid in virus survival and propagation. While both type I and II interferons (IFNs) have been associated with increased bacterial superinfection (BSI) susceptibility, we found that in some cases type I IFNs can be beneficial for BSI outcome. Specifically, we have shown that antagonism of the type I IFN response during infection by some IAVs can lead to the development of deadly BSI. The nonstructural protein 1 (NS1) from IAV is well known for manipulating host type I IFN responses, but the viral proteins mediating BSI severity remain unknown. In this study, we demonstrate that the PDZ-binding motif (PDZ-bm) of the NS1 C-terminal region from mouse-adapted A/Puerto Rico/8/34-H1N1 (PR8) IAV dictates BSI susceptibility through regulation of IFN-α/ß production. Deletion of the NS1 PDZ-bm from PR8 IAV (PR8-TRUNC) resulted in 100% survival and decreased bacterial burden in superinfected mice compared with 0% survival in mice superinfected after PR8 infection. This reduction in BSI susceptibility after infection with PR8-TRUNC was due to the presence of IFN-ß, as protection from BSI was lost in Ifn-ß-/- mice, resembling BSI during PR8 infection. PDZ-bm in PR8-infected mice inhibited the production of IFN-ß posttranscriptionally, and both delayed and reduced expression of the tunable interferon-stimulated genes. Finally, a similar lack of BSI susceptibility, due to the presence of IFN-ß on day 7 post-IAV infection, was also observed after infection of mice with A/TX98-H3N2 virus that naturally lacks a PDZ-bm in NS1, indicating that this mechanism of BSI regulation by NS1 PDZ-bm may not be restricted to PR8 IAV. These results demonstrate that the NS1 C-terminal PDZ-bm, like the one present in PR8 IAV, is involved in controlling susceptibility to BSI through the regulation of IFN-ß, providing new mechanisms for NS1-mediated manipulation of host immunity and BSI severity.

IFNAR2 Is Required for Anti-influenza Immunity and Alters Susceptibility to Post-influenza Bacterial Superinfections.

Influenza virus infections particularly when followed by bacterial superinfections (BSI) result in significant morbidities and mortalities especially during influenza pandemics. Type I interferons (IFNs) regulate both anti-influenza immunity and host susceptibility to subsequent BSIs. These type I IFNs consisting of, among others, 14 IFN-α's and a single IFN-ß, are recognized by and signal through the heterodimeric type I IFN receptor (IFNAR) comprised of IFNAR1 and IFNAR2. However, the individual receptor subunits can bind IFN-ß or IFN-α's independently of each other and induce distinct signaling. The role of type I IFN signaling in regulating host susceptibility to both viral infections and BSI has been only examined with respect to IFNAR1 deficiency. Here, we demonstrate that despite some redundancies, IFNAR1 and IFNAR2 have distinct roles in regulating both anti-influenza A virus (IAV) immunity and in shaping host susceptibility to subsequent BSI caused by S. aureus. We found IFNAR2 to be critical for anti-viral immunity. In contrast to Ifnar1-/- mice, IAV-infected Ifnar2-/- mice displayed both increased and accelerated morbidity and mortality compared to WT mice. Furthermore, unlike IFNAR1, IFNAR2 was sufficient to generate protection from lethal IAV infection when stimulated with IFN-ß. With regards to BSI, unlike what we found previously in Ifnar1-/- mice, Ifnar2-/- mice were not susceptible to BSI induced on day 3 post-IAV, even though absence of IFNAR2 resulted in increased viral burden and an increased inflammatory environment. The Ifnar2-/- mice similar to what we previously found in Ifnar1-/- mice were less susceptible than WT mice to BSI induced on day 7 post-IAV, indicating that signaling through a complete receptor increases BSI susceptibility late during clinical IAV infection. Thus, our results support a role for IFNAR2 in induction of anti-IAV immune responses that are involved in altering host susceptibility to BSI and are essential for decreasing the morbidity and mortality associated with IAV infection. These results begin to elucidate some of the mechanisms involved in how the individual IFNAR subunits shape the anti-viral immune response. Moreover, our results highlight the importance of examining the contributions of entire receptors, as individual subunits can induce distinct outcomes as shown here.

Development of a murine model of lymph node metastases suitable for immunotoxicity studies.

INTRODUCTION: Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice. METHODS: SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated. RESULTS: SCC VII cells express RAE-1, an NKG2D ligand, and were sensitive to natural killer (NK) cells in vitro. QDs were stable in SCC VII cells and showed no evidence of toxicity to the cells. In vivo, confocal microscopy showed that QD-labeled SCC VII cells could migrate to the draining node and microfluorimetry showed progressive traffic of QDs to the node. There was no evidence of systemic toxicity of QDs. Primary immunosuppression in SCID and SCID-beige mice and treatment of normal mice with immunosuppressive agents (anti-asialoGM1 and cyclophosphamide) can enhance traffic of QDs and/or metastases to the draining lymph node. In contrast, cyclosporine had no effect on traffic or metastases. CONCLUSION: This model of local invasion and metastases may be useful in immunotoxicology for identifying and characterizing the hazard posed by selective immunosuppressive drugs.

Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Statistical algorithm for assuring similar efficiency in standards and samples for absolute quantification by real-time reverse transcription polymerase chain reaction.

Reverse transcription (RT) followed by the polymerase chain reaction (PCR) is the method of choice for quantifying rare transcripts in biological samples. A key assumption underlying the absolute quantification of transcripts is similar amplification efficiencies of all external standards and samples. However, efficiencies can vary between individual reactions, a problem that can be magnified when quantifying transcripts of low abundance. Here, an algorithm to assure that calibration standards and samples meet the assumption of similar amplification efficiencies underlying absolute quantification is presented. Individual reaction efficiency is estimated by fitting an exponential growth model to the fluorescence data in the exponential phase of the reaction. Next, reactions of standards with outlying estimates of amplification rates are eliminated using the boxplot outlier detection rule. Then, estimates of amplification rates of outlier-free standards are employed to define exact tolerance intervals, which are used to eliminate kinetic outliers from test samples. This algorithm was employed to eliminate kinetic outliers prior to defining the baseline expression of guanylyl cyclase C mRNA, a marker for colorectal cancer, in blood of healthy volunteers. These studies demonstrate that elimination of kinetic outliers from calibration standards and test samples improves the accuracy of absolute transcript quantification by RT-PCR.