Search

Objective: To develop recommendations for pediatric shared decision-making (SDM)., Methods: We conducted a Delphi method study from 2020 to 2021 with an international panel (n = 21) of clinicians, researchers, and parents with expertise in pediatric SDM. We conducted semistructured interviews to identify the key processes of pediatric SDM. We coded the interviews using content analysis and developed a questionnaire on the potential processes of pediatric SDM. Using a Likert scale, panelists evaluated each process twice, once for simple decisions and once for complex decisions. Panelists were provided with a summary of the results and evaluated each process again. The processes that were agreed on for simple and complex decisions were reported as "fundamental processes." The processes that were agreed on for complex decisions were reported as "additional processes.", Results: A total of 79 recommendations were developed, including 29 fundamental processes and 14 additional processes for complex decisions. A recurring theme was the importance of personalizing the decision-making process. For example, the panel recommended that physicians should assess the family and child's desired roles in the decision-making process, assess their desired level of directiveness, and elicit and clarify their values, preferences, and goals. The panel also disagreed on several subprocesses, such as how to determine the child's role and the appropriate level of directiveness., Conclusions: An international expert panel developed recommendations for pediatric SDM for both simple and complex decisions. The recommendations highlight the importance of personalizing the decision-making process., (Copyright © 2022 by the American Academy of Pediatrics.)

Modifying light fields at the single-photon level is a key challenge for upcoming quantum technologies and can be realized in a scalable manner through integrated quantum photonics. Laser-written diamond photonics offers 3D fabrication capabilities and large mode-field diameters matched to fiber optic technology, though limiting the cooperativity at the single-emitter level. To realize large coupling efficiencies, we combine excitation of single shallow-implanted silicon vacancy centers via high numerical aperture optics with detection assisted by laser-written type-II waveguides. We demonstrate single-emitter extinction measurements with a cooperativity of 0.0050 and a relative beta factor of 13%. The transmission of resonant photons reveals single-photon subtraction from a quasi-coherent field resulting in super-Poissonian light statistics. Our architecture enables light field engineering in an integrated design on the single quantum level although the intrinsic cooperativity is low. Laser-written structures can be fabricated in three dimensions and with a natural connectivity to optical fiber arrays., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

High spatial frequency laser induced periodic surface structure (HSFL) morphology induced by femtosecond laser with 230 fs pulse duration, 250 kHz repetition rate at 1030 nm wavelength on CVD diamond surface is investigated and discussed. The spatial modification was characterized and analyzed by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and 2D-Fast Fourier Transform (2D-FFT). We studied the effect of pulse number and laser power on the spatial development of nanostructures, and also deduced the impact of thermal accumulation effect on their morphology. A generalized plasmonic model has been used to follow the optical evolution of the irradiated surface and to determine the periodic value of the nanostructures. We suggest that non-thermal melting and plasmonic excitation are the main processes responsible for the formation of HSFL-type nanostructures.

Micro-drilling transparent dielectric materials by using non-diffracting beams impinging orthogonally to the sample can be performed without scanning the beam position along the sample thickness. In this work, the laser micromachining process, based on the combination of picosecond pulsed Bessel beams with the trepanning technique, is applied to different transparent materials. We show the possibility to create through-apertures with diameter on the order of tens of micrometers, on dielectric samples with different thermal and mechanical characteristics as well as different thicknesses ranging from two hundred to five hundred micrometers. Advantages and drawbacks of the application of this technique to different materials such as glass, polymer, or diamond are highlighted by analyzing the features, the morphology, and the aspect-ratio of the through-holes generated. Alternative Bessel beam drilling configurations, and the possibility of optimization of the quality of the aperture at the output sample/air interface is also discussed in the case of glass.

An all-diamond photonic circuit was implemented by integrating a diamond microsphere with a femtosecond-laser-written bulk diamond waveguide. The near surface waveguide was fabricated by exploiting the Type II fabrication method to achieve stress-induced waveguiding. Transverse electrically and transverse magnetically polarized light from a tunable laser operating in the near-infrared region was injected into the diamond waveguide, which when coupled to the diamond microsphere showed whispering-gallery modes with a spacing of 0.33 nm and high-quality factors of 10 5 . By carefully engineering these high-quality factor resonances, and further exploiting the properties of existing nitrogen-vacancy centers in diamond microspheres and diamond waveguides in such configurations, it should be possible to realize filtering, sensing and nonlinear optical applications in integrated diamond photonics.

In recent years, there has been significant research on integrated microfluidic devices. Microfluidics offer an advantageous platform for the parallel laminar flow of adjacent solvents of potential use in modern chemistry and biology. To reach that aim, we worked towards the realization of a buried microfluidic Lab-on-a-Chip which enables the separation of the two components by exploiting the non-mixing properties of laminar flow. To fabricate the aforementioned chip, we employed a femtosecond laser irradiation technique followed by chemical etching. To optimize the configuration of the chip, several geometrical and structural parameters were taken into account. The diffusive mass transfer between the two fluids was estimated and the optimal chip configuration for low diffusion rate of the components was defined., Competing Interests: The authors declare no conflict of interest.

We investigate the effect of ultrafast laser surface machining on a monocrystalline synthetic diamond sample by means of pulsed Bessel beams. We discuss the differences of the trench-like microstructures generated in various experimental conditions, by varying the beam cone angle, the energy and pulse duration, and we present a brief comparison of the results with those obtained with the same technique on a sapphire sample. In diamond, we obtain V-shaped trenches whose surface width varies with the cone angle, and which are featured by micrometer sized channels having depths in the range of 10-20 μm. By laser writing crossed trenches we are also able to create and tailor on the diamond surface pillar-like or tip-like microstructures potentially interesting for large surface functionalization, cells capturing and biosensing.

Diamond's nitrogen vacancy (NV) center is an optically active defect with long spin coherence times, showing great potential for both efficient nanoscale magnetometry and quantum information processing schemes. Recently, both the formation of buried 3D optical waveguides and high-quality single NVs in diamond were demonstrated using the versatile femtosecond laser-writing technique. However, until now, combining these technologies has been an outstanding challenge. In this Letter, we fabricate laser-written photonic waveguides in quantum grade diamond which are aligned to within micron resolution to single laser-written NVs, enabling an integrated platform providing deterministically positioned waveguide-coupled NVs. This fabrication technology opens the way toward on-chip optical routing of single photons between NVs and optically integrated spin-based sensing.

We report on the coupling of whispering gallery modes in a 500-μm-radius silicon microsphere to a femtosecond-laser-inscribed glass optical waveguide. The shallow glass waveguide with a large mode field diameter in the near-infrared is written at a depth of 25 μm below the glass surface, resulting in a high excitation impact parameter of 525 μm for the microsphere. The excited whispering gallery modes of the silicon microsphere have quality factors of approximately 10 5 in the 90° elastic scattering and 0° transmission. Integration of such spherical silicon microresonators on femtosecond-laser-inscribed glass waveguides is promising for photonic communication, computation, and sensing applications.

Cognitive photonic networks are researched to efficiently solve computationally hard problems. Flexible fabrication techniques for the implementation of such networks into compact and scalable chips are desirable for the study of new optical computing schemes and algorithm optimization. Here we demonstrate a femtosecond laser-written optical oracle based on cascaded directional couplers in glass, for the solution of the Hamiltonian path problem. By interrogating the integrated photonic chip with ultrashort laser pulses, we were able to distinguish the different paths traveled by light pulses, and thus infer the existence or the absence of the Hamiltonian path in the network by using an optical correlator. This work proves that graph theory problems may be easily implemented in integrated photonic networks, down scaling the net size and speeding up execution times.

Femtosecond laser writing is applied to form Bragg grating waveguides in the diamond bulk. Type II waveguides are integrated with a single pulse point-by-point periodic laser modification positioned toward the edge of the waveguide core. These photonic devices, operating in the telecommunications band, allow for simultaneous optical waveguiding and narrowband reflection from a fourth-order grating. This fabrication technology opens the way toward advanced 3D photonic networks in diamond for a range of applications.

Diamond is a promising platform for sensing and quantum processing owing to the remarkable properties of the nitrogen-vacancy (NV) impurity. The electrons of the NV center, largely localized at the vacancy site, combine to form a spin triplet, which can be polarized with 532 nm laser light, even at room temperature. The NV's states are isolated from environmental perturbations making their spin coherence comparable to trapped ions. An important breakthrough would be in connecting, using waveguides, multiple diamond NVs together optically. However, still lacking is an efficient photonic fabrication method for diamond akin to the photolithographic methods that have revolutionized silicon photonics. Here, we report the first demonstration of three dimensional buried optical waveguides in diamond, inscribed by focused femtosecond high repetition rate laser pulses. Within the waveguides, high quality NV properties are observed, making them promising for integrated magnetometer or quantum information systems on a diamond chip.

Influenza and pneumonia are leading causes of death in elderly populations. With age, there is an increased inflammatory response and slower viral clearance during influenza infection which increases the risk of extended illness and mortality. Here we employ a preclinical murine model of influenza infection to examine the protective capacity of vaccination with influenza nucleoprotein (NP). While NP vaccination reduces influenza-induced lung inflammation in young mice, aged mice do not show this reduction, but are protected from influenza-induced mortality. Aged mice do make a significant amount of NP-specific IgG and adoptive transfer experiments show that NP antibody can protect from death but cannot reduce lung inflammation. Furthermore, young but not aged vaccinated mice generate significant numbers of NP-specific T cells following subsequent infection and few of these T cells are found in aged lungs early during infection. Importantly, aged CD4 T cells have a propensity to differentiate towards a T follicular helper (Tfh) phenotype rather than a T helper 1 (Th1) phenotype that predominates in the young. Since Th1 cells are important in viral clearance, reduced Th1 differentiation in the aged is critical and could account for some or all of the age-related differences in vaccine responses and infection resolution., Competing Interests: CONFLICTS OF INTERESTS The authors have no competing interests in relation to the work described in this manuscript.

An optical realization of the damped quantum oscillator, based on transverse light dynamics in an optical resonator with slowly-moving mirrors, is theoretically suggested. The optical resonator setting provides a simple implementation of the time-dependent Caldirola-Kanai Hamiltonian of the dissipative quantum oscillator and enables the visualization of the effects of damped oscillations in the classical (ray optics) limit and wave packet collapse in the quantum (wave optics) regime.

We report on a strong cross migration of ions in a Tellurite (Te) based glass to form waveguides using a high repetition rate femtosecond laser. The tellurite glass matrix was modified using oxides of P, Na and Zn elements of which Te and Na ions play an important role to form waveguides upon laser irradiation. Tellurium was observed to migrate causing a positive index change zone whereas sodium cross migrates to the tellurium deficient zone forming a relatively low index change region. We have used micro-Raman analysis to scan across the waveguide cross-section to understand the state of the glass network and the relation between ion migration and glass densification for waveguiding. We have found that there is an increase in TeO3 units and reduction of TeO4 units in the Te rich zones enabling densification. This work will help guide the new commercial glass manufacturing industries that aim at producing mid-infrared transparent glasses like tellurite, tellurides and chalcogenides for the production of waveguide based devices.

Two-photon polymerization technology has been used to fabricate submicrometer three-dimensional (3D) structures using a new polyfunctional perfluoropolyether-based resist, which is a polymer intrinsically hydrophobic and chemically resistant. The fluorinated resist was designed and synthesized in this work and successfully employed to fabricate woodpile structures in various experimental conditions. This is the first demonstration of the capability to fabricate hydrophobic and chemically resistant 3D structures with submicrometer resolution and arbitrary geometry.

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cell/extracellular matrix interaction. We applied femtosecond laser two-photon polymerization (2PP) to fabricate three-dimensional (3-D) microscaffolds, or "niches", using a hybrid organic-inorganic photoresist called SZ2080. The niches, of sizes fitting in a volume of 100×100×100 μm(3), were made by an external containment grid of horizontal parallel elements and by an internal 3-D lattice. We developed two niche heights, 20 and 80-100 μm, and four lattice pore dimensions (10, 20, 30 μm and graded). We used primary rat mesenchymal stem cells (MSCs) to study cell viability, migration and proliferation in the niches, up to 6 culture days. MSCs preferentially stayed on/in the structures once they ran into them through random migration from the surrounding flat surface, invaded those with a lattice pore dimension greater than 10 μm, and adhered to the internal lattice while the cell nuclei acquired a roundish morphology. In the niches, the highest MSC density was found in those areas where proliferation was observed, corresponding to the regions where the scaffold surface density available for cell adhesion was highest. The microgeometry inducing the highest cell density was 20 μm high with graded pores, in which cell invasion was favored in the central region of large porosity and cell adhesion was favored in the lateral regions of high scaffold surface density. Cell density in the niches, 17±6 cells/(100×100 μm(2)), did not significantly differ from that of the flat surface colonies. This implies that MSCs spontaneously homed and established colonies within the 3-D niches. This study brings to light the crucial role played by the niche 3-D geometry on MSC colonization in culture, with potential implications for the design of biomaterial scaffolds for synthetic niche engineering., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Influenza causes >250,000 deaths annually in the industrialized world, and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. In this study, we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either Gram-positive Streptococcus pneumoniae or Gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and Abs contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas Abs to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that nonneutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.

Fully functional T cells are necessary for the maintenance of protective immunity during chronic infections. However, activated T cells often undergo apoptosis or exhaustion upon chronic stimulation mediated by Ag or inflammation. T cell attrition can be compensated for by the production of thymus-derived T cells, although the new naive T cells must undergo T cell priming and differentiation under conditions different from those encountered during acute infection. We used a murine model of Mycobacterium tuberculosis infection to address how the activation and differentiation of new thymic emigrants is affected by chronic inflammation, as well as whether the newly developed effector T cells help to maintain peripheral T cell responses. Although new thymic emigrants contributed to the peripheral T cell response early during acute M. tuberculosis infection, the relative contribution of new effector T cells to the peripheral CD4 and CD8 T cell pools declined during chronic infection. The decline in new T cell recruitment was a consequence of quantitative and/or qualitative changes in Ag presentation, because during chronic infection both the priming and expansion of naive T cells were inefficient. Thus, although thymic tolerance is not a major factor that limits protective T cell responses, the chronic environment does not efficiently support naive T cell priming and accumulation during M. tuberculosis infection. These studies support our previous findings that long-term protective T cell responses can be maintained indefinitely in the periphery, but also suggest that the perturbation of homeostasis during chronic inflammatory responses may elicit immune pathology mediated by new T cells.

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T-cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T-cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T-cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T-cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations., (© 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

This paper provides an overview of femtosecond laser microfabrication in polymeric materials, with emphasis on lab-on-chip applications. Due to the nonlinear interaction of femtosecond laser pulses with polymers, laser-induced modifications are localized to the focal volume, enabling high resolution patterning in 3D. Femtosecond laser microfabrication offers unmatched versatility in fabricating surface microchannels and diffractive optics by means of laser ablation, buried optical waveguides and micro-optics through refractive index modification and complex 3D microstructures in photoresists by two-photon polymerization. Femtosecond laser microfabrication technology opens the door to fabricating integrated lab-on-chip devices with a single tool., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to determine the dose of weekly oral topotecan that allows safe administration and to evaluate the pharmacokinetics of this dose in patients with recurrent gynecologic malignancies. The first cohort of patients received oral topotecan 6 mg/week administered orally on days 1, 8, and 15 of a 28-day regimen. A standard 3+3 dose-escalating phase design was used for dose levels II-V (8, 10, 12 and 14 mg/week). Toxicity was scored according to the Common Terminology Criteria for Adverse Events. Cumulative toxicity was summarized in the 6-12 mg/week combined cohort and 14 mg/week cohort separately. Pharmacokinetic samples were obtained for day 1, cycle 1 only in the expansion cohort (dose level V). Twenty-five patients received a total of 88 cycles of therapy. Hematologic toxicities of grade 3 (6-12 mg dose) were neutropenia (25%) and anemia (8.3%). Gastrointestinal toxicities of grade 3 were diarrhea (16.7%) and obstruction (8.3%, disease-related). Grade 3 or 4 (14 mg/week) hematologic toxicities consisted of neutropenia (38.5%), platelets (15.4%), anemia (15.4%), infection with neutropenia (7.7%), and thrombosis (7.7%). Gastrointestinal toxicities of grade 3 were diarrhea (7.7%), obstruction (7.7%), and vomiting (7.7%). One patient died secondary to neutropenic sepsis. One patient (4%; 95% confidence interval: 2.1, 22.3) showed a partial response and five patients (20%; 95% confidence interval: 7.6, 41.3) had stable disease. An oral topotecan dose of 14 mg/week for 3 consecutive weeks out of 4 is mostly associated with acceptable toxicities and may be considered for use in future single-agent phase II trials.

Three-dimensional material microstructuring by femtosecond laser-induced two-photon polymerization is emerging as an important tool in biomedicine. During two-photon polymerization, a tightly focused femtosecond laser pulse induces a crosslinking photoreaction in the polymer confined within the focal volume. As a rapid-prototyping technique, two-photon polymerization enables the fabrication of truly arbitrary three-dimensional micro- and nano-structures directly from computer models, with a spatial resolution down to 100 nm. In this review, we discuss the fundamentals, experimental methods, and materials used for two-photon polymerization; in addition, we present some applications of this technology related to microfluidics and to biomaterial scaffolds for tissue engineering and regenerative medicine.

Influenza virus infection is considered a major worldwide public health problem. Seasonal infections with the most common influenza virus strains (e.g., H1N1) can usually be resolved, but they still cause a high rate of mortality. The factors that influence the outcome of the infection remain unclear. Here, we show that deficiency of interleukin (IL)-6 or IL-6 receptor is sufficient for normally sublethal doses of H1N1 influenza A virus to cause death in mice. IL-6 is necessary for resolution of influenza infection by protecting neutrophils from virus-induced death in the lung and by promoting neutrophil-mediated viral clearance. Loss of IL-6 results in persistence of the influenza virus in the lung leading to pronounced lung damage and, ultimately, death. Thus, we demonstrate that IL-6 is a vital innate immune cytokine in providing protection against influenza A infection. Genetic or environmental factors that impair IL-6 production or signaling could increase mortality to influenza virus infection.

We report on the integration of a size-based three-dimensional filter, with micrometre-sized pores, in a commercial microfluidic chip. The filter is fabricated inside an already sealed microfluidic channel using the unique capabilities of two-photon polymerization. This direct-write technique enables integration of the filter by post-processing in a chip that has been fabricated by standard technologies. The filter is located at the intersection of two channels in order to control the amount of flow passing through the filter. Tests with a suspension of 3 μm polystyrene spheres in a Rhodamine 6G solution show that 100% of the spheres are stopped, while the fluorescent molecules are transmitted through the filter. We demonstrate operation up to a period of 25 minutes without any evidence of clogging. Preliminary validation of the device for plasma separation from whole blood is shown. Moreover, the filter can be cleaned and reused by reversing the flow.

Influenza A infection induces a massive inflammatory response in the lungs that leads to significant illness and increases the susceptibility to secondary bacterial pneumonia. The most efficient way to prevent influenza infection is through vaccination. While inactivated vaccines induce protective levels of serum antibodies to influenza hemaglutinin (HA) and neuraminidase (NA) surface proteins, these are strain specific and offer little protection against heterosubtypic influenza viruses. In contrast, live attenuated influenza vaccines (LAIVs) induce a T cell response in addition to antibody responses against HA and NA surface proteins. Importantly, LAIV vaccination induces a response in a mouse model that protects against illness due to heterosubtypic influenza strains. While it is not completely clear what is the mechanism of action of LAIV heterosubtypic protection in humans, it has been shown that LAIV induces heterosubtypic protection in mice that is dependent upon a Type 1 immune response and requires CD8 T cells. In this study, we show that LAIV-induced immunity leads to significantly reduced viral titers and inflammatory responses in the lungs of mice following heterosubtypic infection. Not only are viral titers reduced in LAIV vaccinated mice, the amounts of inflammatory cytokines and chemokines in lung tissue are significantly lower. Additionally, we show that LAIV vaccination of healthy adults also induces a robust Type 1 memory response including the production of chemokines and cytokines involved in T cell activation and recruitment. Thus, our results indicate that LAIV vaccination functions by inducing immune memory which can act to modulate the immune response to subsequent heterosubtypic challenge by influencing both innate and adaptive responses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We demonstrate that hydrophobic areas formed by femtosecond laser irradiation on poly(methyl methacrylate) (PMMA) and polystyrene (PS) polymer substrates can be faithfully replicated on samples of the same material via a solvent-resistant perfluoropolyether (PFPE) elastomer mold. The replicated PMMA and PS samples show nearly identical micro-nanoscale topography and hydrophobic wetting characteristics as the laser-patterned master substrates. This work combines the femtosecond laser capability of spatially tailoring the wettability with a high-resolution parallel replication method, offering the potential for the efficient production of microfluidic devices with selectively tailored flow behavior., (© 2011 American Chemical Society)

We report on the fabrication of binary Fresnel lenses by femtosecond laser surface ablation of poly(methyl methacrylate) (PMMA) substrates. Tight focusing of the laser pulses produced a minimum ablated feature size of 600 nm, enabling the creation of lenses with numerical apertures as high as 0.5 and focal lengths ranging from 500 µm to 5 mm. A precise control of the ablation depth allowed the achievement of a 30% focusing efficiency, close to the maximum theoretical value for this kind of lenses.

We report on the first demonstration of an optical waveguide amplifier in phospho-tellurite glass providing net gain at 1.5 μm. The device was fabricated using a high repetition rate femtosecond laser and exhibited internal gain across 100-nm bandwidth covering the entire C + L telecom bands.

The effects of femtosecond laser ablation on the physical and chemical properties at the surface of poly methylmethacrylate (PMMA) were studied. Femtosecond laser microfabrication caused the initially wetting behavior of PMMA to become nonwetting, mainly because of the laser-induced surface porosity at the submicroscale. Static and dynamic contact angle measurements along with morphological characterization revealed that after the laser irradiation, the system lies in an intermediate regime between those theorized by Wenzel and Cassie-Baxter. Spectroscopic analysis did not evidence any significant variation in the chemical properties of the processed polymeric surfaces.

The biological outcome of receptor-mediated signalling often depends on the duration of engagement. Because CD40 signalling is controlled by the regulated expression of its ligand, CD154, the mechanisms that regulate CD154 expression probably determine the strength and duration of CD40 signalling. Here, we demonstrate that CD154 expression on the surface of mouse CD4 T cells can be separated into an early phase, occurring between 0 and 24 hr after T-cell activation, and a later extended phase, occurring after 24 hr. The early phase of CD154 expression did not require costimulation and was probably influenced by the strength of T-cell receptor (TCR) signalling alone. However, later CD154 expression was highly dependent on costimulation through either CD28 or inducible costimulator (ICOS). Although CD28 signalling interleukin (IL)-2 secretion, ICOS not, suggesting that costimulation enhance CD154 expression independently of IL-2 production. In fact, anti-CD28 treatment could still induce late-phase CD154 on anti-CD3-stimulated CD4 T cells expressing a mutated form of CD28 that not lead to the induction of IL-2. However, this CD154 induction was somewhat weaker than that of wild-type CD28-expressing cells, suggesting that direct signalling and IL-2-mediated signalling co-operatively responsible for the levels of CD154 induced by CD28. Finally, we show that the second phase of CD154 expression negatively regulated B-cell terminal differentiation and antibody secretion. These results demonstrate that TCR signalling and costimulation each regulate different phases of CD154 expression and control the biological outcome of CD40 signalling on B cells.

The metabolism of (177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-(4-aminobenzoyl)-QWAVGHLM-NH(2)), a radiotherapeutic compound in clinical development that binds to GRP and NMB receptors, was studied in vitro (mouse, rat and human plasma, mouse kidney homogenate) and in vivo (by analysis of mouse and rat plasma and urine following IV injection of (177)Lu-AMBA). The primary metabolites were Lu-DO3A-CH(2)CO-G-Abz4-R, where R = -Q-OH (A), -QW-OH (B), and -QWAVGH-OH (C). Minor amounts of (D) where R = -QWAVGHLM-OH and (E) -QWAVGHL-OH were also observed. Clearance of (177)Lu-AMBA and of radioactivity from mouse and rat blood was rapid in vivo. In mouse and rat urine, only metabolites Lu-A and Lu-B were found-no parent drug was excreted. Unmetalated ligands and (nat)Lu and (177)Lu complexes for Lu-AMBA metabolites A-E were synthesized, characterized by HPLC and MS, and used to perform in vitro competition and direct binding studies on GRP receptor-positive PC-3 (human prostate) cancer cells. Biodistribution studies with (177)Lu-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact (177)Lu-AMBA. IC(50) values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against (177)Lu-AMBA. No direct binding to PC-3 cells was observed with (177)Lu-labeled A-C, confirming IC(50) results. (177)Lu-labeled metabolites A-E showed no uptake in GRP-receptor positive tumor or pancreas in PC-3 tumor bearing mice. All metabolites were rapidly excreted via the renal route (approximately 78-87%) within 1 h. These results demonstrate that the tumor uptake observed with (177)Lu-AMBA is due to parent drug and not due to any of its identified metabolites.

Age-related declines in humoral responses contribute to the reduced efficacy of vaccines in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in CD4 T cell function contribute significantly to the reduced humoral responses observed with aging, resulting in reduced B cell expansion and differentiation as well as reduced IgG production. In this current study, we show that the helper function of aged CD4 T cells can be enhanced using a TLR-binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. The helper function of aged CD4 T cells was also enhanced when PI cytokines were added during in vitro CD4 effector generation. Enhanced helper activity resulted in improved expansion and differentiation of B cells and affinity maturation of IgG. PI cytokines also induced significant production of effector cytokines, including IL-4, IFN-gamma, IL-17, and IL-21, by both young and aged CD4 T cells. Importantly, we also show that proinflammatory adjuvants can significantly enhance the humoral response in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding CD4 T cells.

Interleukin (IL) 6 is a proinflammtory cytokine produced by antigen-presenting cells and nonhematopoietic cells in response to external stimuli. It was initially identified as a B cell growth factor and inducer of plasma cell differentiation in vitro and plays an important role in antibody production and class switching in vivo. However, it is not clear whether IL-6 directly affects B cells or acts through other mechanisms. We show that IL-6 is sufficient and necessary to induce IL-21 production by naive and memory CD4(+) T cells upon T cell receptor stimulation. IL-21 production by CD4(+) T cells is required for IL-6 to promote B cell antibody production in vitro. Moreover, administration of IL-6 with inactive influenza virus enhances virus-specific antibody production, and importantly, this effect is dependent on IL-21. Thus, IL-6 promotes antibody production by promoting the B cell helper capabilities of CD4(+) T cells through increased IL-21 production. IL-6 could therefore be a potential coadjuvant to enhance humoral immunity.

Understanding how aging impacts the function of memory CD4 T cells is critical for designing effective vaccines. Our studies show that immunological memory generated during youth functions well into old age, whereas that generated later in life functions poorly. This is the result of declines in the function of naive CD4 T cells from aged individuals and contributes to reduced efficacy of vaccines in the elderly. To begin to identify the cause of this defect, we examined the function of memory T cells generated from bone marrow precursor cells (BMPC) from young or aged mice in young hosts. In two different models, memory cells derived from young and aged BMPC exhibit good ex vivo and in vivo function. Importantly, memory CD4 T cells generated from aged BMPC exhibit potent cognate helper function for humoral responses, which are critical for effective immunization. These results indicate that there are no apparent age-related intrinsic defects in BMPC with regards to generation of functional memory T cells.

A single-step fast-writing method of burst ultrafast laser modification was applied to form a mesh network of multi-wavelength Bragg grating waveguides in bulk fused silica glass. Strain-optic and thermo-optic responses of the laser-written internal sensors are reported for the first time. A dual planar layout provided independent temperature- and strain-compensated characterization of temperature and strain distribution with coarse spatial resolution. The grating responses were thermally stable to 500 masculineC. To our best knowledge, the grating network represents the first demonstration of 3D distributed optical sensing network in a bulk transparent medium. Such 3D grating networks open new directions for strain and temperature sensing in optical circuits, optofluidic, MEMS or lab-on-a-chip microsystems, actuators, and windows and other large display or civil structures.

A femtosecond fiber laser was applied to fabricate broadband directional couplers inside bulk glass for general power splitting application in the 1250 to 1650-nm wavelength telecom spectrum. The broadband response was optimized over the 400-nm bandwidth by tailoring the coupling strength and the waveguide interaction length to balance the differing wavelength dependence of the straight interaction and bent transition regions. High spatial finesse of the femtosecond-laser writing technique enabled close placement (approxiamtely 6 microm) of adjacent waveguides that underpinned the wavelength-flattened broadband response at any coupling ratio in the 0% to 100% range. The spectral responses were well-represented by coupled mode theory, permitting simple design and implementation of broadband couplers for bulk 3D optical circuit integration.

A variable (0.2 to 5 MHz) repetition rate femtosecond laser was applied to delineate the role of thermal diffusion and heat accumulation effects in forming low-loss optical waveguides in borosilicate glass across a broad range of laser exposure conditions. For the first time, a smooth transition from diffusion-only transport at 200 kHz repetition rate to strong heat accumulation effects at 0.5 to 2 MHz was observed and shown to drive significant variations in waveguide morphology, with rapidly increasing waveguide diameter that accurately followed a simple thermal diffusion model over all exposure variables tested. Amongst these strong thermal trends, a common exposure window of 200 mW average power and approximately 15-mm/s scan speed was discovered across the range of 200 kHz to 2 MHz repetition rates for minimizing insertion loss despite a 10-fold drop in laser pulse energy. Waveguide morphology and thermal modeling indicate that strong thermal diffusion effects at 200 kHz give way to a weak heat accumulation effect at approximately 1 microJ pulse energy for generating low loss waveguides, while stronger heat accumulation effects above 1-MHz repetition rate offered overall superior guiding. A comprehensive characterization of waveguide properties is presented for laser writing in the thermal diffusion and heat accumulation regimes. The waveguides are shown to be thermally stable up to 800 degrees C and can be written in a convenient 520 microm depth range with low spherical aberration.

For the first time femtosecond-laser writing has inscribed low-loss optical waveguides in Schott BK7 glass, a commercially important type of borosilicate widely used in optical applications. The use of a variable repetition rate laser enabled the identification of a narrow processing window at 1 MHz repetition rate with optimal waveguides exhibiting propagation losses of 0.3 dB/cm and efficient mode matching to standard optical fibers at a 1550 nm wavelength. The waveguides were characterized by complementary phase contrast and optical transmission microscopy, identifying a micrometer-sized guiding region within a larger complex structure of both positive and negative refractive index variations.

For the first time to our knowledge, high-strength (>30 dB) first-order Bragg grating waveguides were fabricated in bulk fused silica glass in a single-scanning step by modulating a high-repetition-rate femtosecond fiber laser with an external acousto-optic modulator. The modulation induced a waveguide segmentation by delivering controlled bursts of laser pulses to define an array of partially overlapped refractive index voxels. With appropriate choice of modulation frequency and sample scanning speed, low loss waveguides could be formed at high writing speeds to yield sharp Bragg spectral resonances tunable over the 1300 to 1550 nm telecom band. Effects of acousto-optic modulation duty cycle on propagation loss and grating strength are characterized. This modulation method offers facile control and integration of multiwavelength Bragg grating devices to enhance overall functionality of optical circuits in three-dimensional geometries.

In contrast to naïve CD4+ T cells, memory CD4+ T cells rapidly express high levels of effector cytokines in response to antigen stimulation. The molecular mechanism for this specific behavior is not well understood. The nuclear factor of activated T cells (NFAT) family of transcription factors plays an important role in the transcription of many cytokine genes. Here we show that memory CD4+ T cells rapidly induce NFAT-mediated transcription upon T cell receptor ligation whereas NFAT activation in naïve CD4+ T cells requires longer periods of stimulation. The difference in kinetics correlates with the low levels of NFATc1 and NFATc2 proteins present in naïve CD4+ T cells and their high levels in memory CD4+ T cells. Accordingly, IL-2 expression requires NFAT activation only in memory CD4+ T cells whereas it is NFAT-independent in naïve CD4+ T cells. Thus, the accumulation of NFATc1 and NFATc2 in memory CD4+ T cells represents a previously uncharacterized regulatory mechanism for the induction of early gene expression after antigen stimulation.

A one-step type II photosensitivity process has been optimized for inscribing strong >30-dB first-order Bragg-gratings during laser formation of buried waveguides in borosilicate glass. Mode profiles, propagation losses, waveguide birefringence and transmission and reflection spectra were systematically studied in the 1550-nm telecom band over a wide range of laser exposure conditions. Low-loss and birefringence-free waveguides are reported for a narrow laser processing window of 1.0 +/- 0.2 ps pulse duration, yielding highly stable Bragg resonances to temperatures up to 500 degrees C.

Interferon-gamma is key in limiting Mycobacterium tuberculosis infection. Here we show that vaccination triggered an accelerated interferon-gamma response by CD4(+) T cells in the lung during subsequent M. tuberculosis infection. Interleukin 23 (IL-23) was essential for the accelerated response, for early cessation of bacterial growth and for establishment of an IL-17-producing CD4(+) T cell population in the lung. The recall response of the IL-17-producing CD4(+) T cell population occurred concurrently with expression of the chemokines CXCL9, CXCL10 and CXCL11. Depletion of IL-17 during challenge reduced the chemokine expression and accumulation of CD4(+) T cells producing interferon-gamma in the lung. We propose that vaccination induces IL-17-producing CD4(+) T cells that populate the lung and, after challenge, trigger the production of chemokines that recruit CD4(+) T cells producing interferon-gamma, which ultimately restrict bacterial growth.

Novel Bragg grating waveguide structures have been fabricated in bulk borosilicate glass through a type II photosensitivity mechanism driven by single femtosecond laser pulses. Low-loss single-mode waveguides and narrow-linewidth Bragg gratings were generated simultaneously by forming an array of refractive index voxels in a single laser scan. Laser pulse duration is shown to significantly affect the grating strength and waveguide loss. Bragg wavelengths, defined by the periodicity of laser-modified volumes, were fully controlled by the sample scan speed to provide resonances anywhere in the 1200-1620 nm telecommunication bands. Four linear Bragg filters with distinct resonant wavelengths are presented that define the first demonstration of laser writing of multiple-wavelength and cascaded Bragg grating waveguides in a single process step.

Unlabelled: Gastrin-releasing peptide receptors (GRP-R) are upregulated in many cancers, including prostate, breast, and lung. We describe a new radiolabeled bombesin (BBN) analog for imaging and systemic radiotherapy that has improved pharmacokinetics (PK) and better retention of radioactivity in the tumor., Methods: DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA) was synthesized and radiolabeled. The human prostate cancer cell line PC-3 was used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Receptor specificity was determined by in vitro autoradiography in human tissues. PK and radiotherapy studies were performed in PC-3 tumor-bearing male nude mice., Results: 177Lu-AMBA has a high affinity for the GRP-R (Kd, 1.02 nmol/L), with a maximum binding capacity (Bmax) of 414 fmol/10(6) cells (2.5 x 10(5) GRP-R/cell). Internalization was similar for 177Lu-AMBA (76.8%), 177Lu-BBN8 (72.9%), and 125I-[Tyr4]-BBN (74.9%). Efflux was markedly lower for 177Lu-AMBA (2.9%) compared with 177Lu-BBN8 (15.9%) and 125I-[Tyr4]-BBN (46.1%). By receptor autoradiography, Lu-AMBA binds specifically to GRP-R (0.8 nmol/L) and to the neuromedin B receptor (NMB-R) (0.9 nmol/L), with no affinity for the bb3 receptor (>1,000 nmol/L). 177Lu-AMBA was renally excreted (55 %ID 1 h [percentage injected dose at 1 h]); tumor uptake at 1 and 24 h was 6.35 %ID/g and 3.39 %ID/g, respectively. One or 2 doses of 177Lu-AMBA (27.75 MBq/dose) significantly prolonged the life span of PC-3 tumor-bearing mice (P < 0.001 and P < 0.0001, respectively) and decreased PC-3 tumor growth rate over controls. When compared using World Health Organization criteria, mice receiving 2 doses versus 1 dose of 177Lu-AMBA demonstrated a shift away from stable/progressive disease toward complete/partial response; by RECIST (Response Evaluation Criteria in Solid Tumors), median survival increased by 36% and time to progression/progression-free survival increased by 65%., Conclusion: 177Lu-AMBA binds with nanomolar affinity to GRP-R and NMB-R, has low retention of radioactivity in kidney, demonstrates a very favorable risk-benefit profile, and is in phase I clinical trials.

A new domain of rapid waveguide writing with non-overlapping pulses of a 1-kHz ultrashort laser is demonstrated to produce low loss waveguides in fused silica glass. This new regime is distinguishable in two ways from traditional approaches in laser waveguide writing. First, an examination of a wide 50-fs to 5-ps range of pulse duration shows the lowest loss waveguides to form in a narrow 1.0 +/- 0.2 ps window that significantly exceeds the 50 - 200 fs duration reported as optimal in other studies. Second, an unusually high scan speed of 1.0 +/- 0.2 mm/s points to a novel Type-II photosensitivity mechanism for generating low-loss refractive index structures. The waveguides comprise of an array of nearly isolated single-pulse interaction volumes that sharply contrast with the high exposures of tens to thousands of overlapping laser pulses typically applied along a slowly moving focal volume. A minimum propagation loss of ~0.2 dB/cm and a slightly asymmetric mode diameter of ~9 mum is reported for 633-nm light. The low loss waveguides fabricated with picosecond pulses enables 3-D photonics circuit fabrication with simpler and lower cost picosecond laser systems.

The kinetics of presentation of influenza virus-derived antigens (Ags), resulting in CD4 T cell effector and memory generation, remains undefined. Naive influenza-specific CD4 T cells were transferred into mice at various times after influenza infection to determine the duration and impact of virus-derived Ag presentation. Ag-specific T cell responses were generated even when the donor T cells were transferred 3-4 wk after viral clearance. Transfer of naive CD4 T cells during early phases of infection resulted in a robust expansion of highly differentiated effectors, which then contracted to a small number of memory T cells. Importantly, T cell transfer during later phases of infection resulted in a modest expansion of effectors with intermediate phenotypes, which were capable of persisting as memory with high efficiency. Thus, distinct stages of pathogen-derived Ag presentation may provide a mechanism by which T cell heterogeneity is generated and diverse memory subsets are maintained.

With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4(+) T-cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen-specific naive T cells from young and aged T-cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)-2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B-cell expansion and differentiation. These age-related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL-2. We also found no difference in B-cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age-related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4(+) T cells from aged individuals.

Using a T cell receptor transgenic (TCR Tg) mouse model, we have shown that TCR Tg CD4 cells from aged mice retain a naive phenotype, but exhibit reduced proliferation and IL-2 production in response to the antigen compared with cells from young mice. We hypothesize that age-related decreases in T cell function may be partly related to the age of the T cells. Because thymic output is decreased with age, peripheral T cells in older individuals are likely to be older than those in younger individuals. To investigate this possibility, we have manipulated the age of CD4 T cells in the periphery of young and aged mice. The production of new T cells was induced by depleting peripheral CD4 T cells or by creating bone marrow chimeras. In both young and aged individuals where we induced the production of new T cells, these newly generated cells exhibited robust responses to antigen ex vivo and in vivo, exhibiting good expansion, IL-2 production, and cognate helper function. Our results suggest that age-related defects in response to antigenic stimulation, in part, are caused by the age of the CD4 T cells.