Measuring the linear optical absorption coefficient by interferometry and the thermal lensing effect: a numerical analysis.

We report on a pump-probe thermal lensing method for measuring the linear absorption coefficient of liquids by using interferometry and numerical analysis. The method is based on interferograms generated when a localized photothermal effect is induced in the sample. The photothermal effect itself is induced by a pump beam impinging on a sample located on-axis of the probe beam, which is one of the paths of a Mach-Zehnder interferometer. A digital camera is employed as the acquisition device allowing the capture and storage of the experimental data. During the experiment, a total of three photographs are taken and stored on a personal computer, and by using an algorithm, the numerical analysis is done. Numerical analysis is subsequently used to calculate the photothermal phase difference and the normalized spatial distribution of the pump beam irradiance. Plotting the phase difference as a function of the spatial distribution of the pump beam produces a linear dependence from which the linear absorption coefficient is obtained. The sensitivity of the method (λ/1500) is validated using ethanol, methanol, and carbon disulfide.

IRF5 and IRF8 modulate the CAL-1 human plasmacytoid dendritic cell line response following TLR9 ligation.

Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFNs) and proinflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) play a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF5 and IRF8 (also known as IFN consensus sequence-binding protein) on CpG-dependent gene expression in the human CAL-1 pDC cell line. This cell line shares many of the phenotypic and functional properties of freshly isolated human pDCs. Results from RNA interference and microarray studies indicate that IRF5 upregulates TLR9-driven gene expression whereas IRF8 downregulates the same genes. Several findings support the conclusion that IRF8 inhibits TLR9-dependent gene expression by directly blocking the activity of IRF5. First, the inhibitory activity of IRF8 is only observed when IRF5 is present. Second, proximity ligation analysis shows that IRF8 and IRF5 colocalize within the cytoplasm of resting human pDCs and cotranslocate to the nucleus after CpG stimulation. Taken together, these findings suggest that IRF5 and IRF8, two transcription factors with opposing functions, control TLR9 signaling in human pDCs.

ß-Defensin 2 and 3 promote the uptake of self or CpG DNA, enhance IFN-α production by human plasmacytoid dendritic cells, and promote inflammation.

Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. In this article, we present a novel role for two potent alarmins, human ß-defensin 2 and 3 (HBD2 and 3), in promoting IFN-α production by human plasmacytoid dendritic cells. We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA-induced IFN-α production in a TLR9-dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal titration calorimetry studies to elucidate the nature of HBD3/CpG complexes demonstrate involvement of enthalpy-driven interactions, in addition to hydrophobic interactions, with the formation of complexes at a molar ratio of 2:1 defensin/CpG. The i.v. administration of HBD3/CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α, and IL-10 in serum, associated with an increased recruitment of APCs in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses to OVA, compared with OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus, the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells, resulting in an enhanced immune response.

IRF-5 and NF-κB p50 co-regulate IFN-ß and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells.

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN-ß) and pro-inflammatory (IL-6) cytokines by CpG-stimulated human pDCs and the human CAL-1 pDC cell line. Results show that interferon regulatory factor-5 (IRF-5) and NF-κB p50 are key co-regulators of IFN-ß and IL-6 expression following TLR9-mediated activation of human pDCs. The nuclear accumulation of IRF-1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF-8 was identified as a novel negative regulator of gene activation in CpG-stimulated pDCs. As variants of IRF-5 and IRF-8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of "K" ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.

Thermal lensing approach based on parabolic approximation and Mach-Zehnder interferometer.

A thermal lensing approach based on parabolic approximation and Mach-Zehnder interferometer for measuring optical absorption and thermal diffusivity coefficients in pure solvents is described in this work. The approach combines the sensitivity of both thermal lensing methods and interferometry techniques. The photothermal effect is induced by a pump laser beam generating localized changes in the refractive index of the sample, which are observed as a shift in phase of the interference pattern. Each interference pattern is recorded by means of a digital camera and stored as digital images as a function of time. The images are then numerically processed to calculate the phase shifting map for a specific time. From each phase shifting map, the experimental phase difference as a function of time is calculated giving a phase-time transient, which is fitted to a mathematical model to estimate the optical absorption and thermal diffusivity of the sample. The experimental results show that the sensitivity is approximately λ/4800 for the minimum phase difference measured.

Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism.

Wnt4 and ß-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of ß-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of ß-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of ß-catenin's helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of ß-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca(2+)-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca(2+) and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca(2+)-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.

Long-Term Efficacy and Safety of Brigatinib in Crizotinib-Refractory ALK+ NSCLC: Final Results of the Phase 1/2 and Randomized Phase 2 (ALTA) Trials.

Introduction: We report brigatinib long-term efficacy and safety from phase 1/2 and phase 2 (ALTA) trials in ALK-rearrangement positive (ALK+) NSCLC. Methods: The phase 1/2 study evaluated brigatinib 30 to 300 mg/d in patients with advanced malignancies. ALTA randomized patients with crizotinib-refractory ALK+ NSCLC to brigatinib 90 mg once daily (arm A) or 180 mg once daily (7-d lead-in at 90 mg; arm B). Results: In the phase 1/2 study, 79 of 137 brigatinib-treated patients had ALK+ NSCLC; 71 were crizotinib pretreated. ALTA randomized 222 patients (n = 112 in arm A; n = 110 in arm B). Median follow-up at phase 1/2 study end (≈5.6 y after last patient enrolled) was 27.7 months; at ALTA study end (≈4.4 y after last patient enrolled), 19.6 months (A) and 28.3 months (B). Among patients with ALK+ NSCLC in the phase 1/2 study, median investigator-assessed progression-free survival (PFS) was 14.5 months (95% confidence interval [CI]: 10.8-21.2); median overall survival was 47.6 months (28.6-not reached). In ALTA, median investigator-assessed PFS was 9.2 months (7.4-11.1) in arm A and 15.6 months (11.1-18.5) in arm B; median independent review committee (IRC)-assessed PFS was 9.9 (7.4-12.8) and 16.7 (11.6-21.4) months, respectively; median overall survival was 25.9 (18.2-45.8) and 40.6 (32.5-not reached) months, respectively. Median intracranial PFS for patients with any brain metastases was 12.8 (9.2-18.4) months in arm A and 18.4 (12.6-23.9) months in arm B. No new safety signals were identified versus previous analyses. Conclusions: Brigatinib exhibited sustained long-term activity and PFS with manageable safety in patients with crizotinib-refractory ALK+ NSCLC.

An array-based method to identify multivalent inhibitors.

Carbohydrate-protein interactions play a critical role in a variety of biological processes, and agonists/antagonists of these interactions are useful as biological probes and therapeutic agents. Most carbohydrate-binding proteins achieve tight binding through formation of a multivalent complex. Therefore, both ligand structure and presentation contribute to recognition. Since there are many potential combinations of structure, spacing, and orientation to consider and the optimal one cannot be predicted, high-throughput approaches for analyzing carbohydrate-protein interactions and designing inhibitors are appealing. In this report, we develop a strategy to vary neoglycoprotein density on a surface of a glycan array. This feature of presentation was combined with variations in glycan structure and glycan density to produce an array with approximately 600 combinations of glycan structure and presentation. The unique array platform allows one to distinguish between different types of multivalent complexes on the array surface. To illustrate the advantages of this format, it was used to rapidly identify multivalent probes for various lectins. The new array was first tested with several plant lectins, including concanavalin A (conA), Vicia villosa isolectin B4 (VVL-B(4)), and Ricinus communis agglutinin (RCA120). Next, it was used to rapidly identify potent multivalent inhibitors of Pseudomonas aeruginosa lectin I (PA-IL), a key protein involved in opportunistic infections of P. aeruginosa , and mouse macrophage galactose-type lectin (mMGL-2), a protein expressed on antigen presenting cells that may be useful as a vaccine targeting receptor. An advantage of the approach is that structural information about the lectin/receptor is not required to obtain a multivalent inhibitor/probe.

Effects of hapten density on the induced antibody repertoire.

Small peptides and oligosaccharides are important antigens for the development of vaccines and the production of monoclonal antibodies. Because of their small size, peptides and oligosaccharides are non-immunogenic on their own and typically must be conjugated to a larger carrier protein to elicit an immune response. Selection of a suitable carrier protein, conjugation method, and hapten density are critical for generating an optimal immune response. We used a glycan array to compare the repertoire of antibodies induced after immunizing with either low or high-density conjugates of the tumor-associated Tn antigen. At high hapten density, a broader range of antibodies was induced, and reactivity to the clustered Tn antigen was observed. In contrast, antibodies induced by the low-density conjugate had narrower reactivity and did not bind the clustered Tn antigen.