Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma

Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3⁺), cytotoxic-T cells (CD8⁺), T-helper cells (CD4⁺), B cells (CD20⁺), macrophages (CD68⁺), mast cells (CD117⁺), mononuclear cells (CD11c⁺), plasma cells, activated-T cells (MUM1⁺), B cells, myeloid cells (PD1⁺) and neutrophilic granulocytes (myeloperoxidase⁺) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1⁺ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.     

Diabetes mellitus induced by low-dose interleukin-2

 Interleukin-2 (IL-2) is a potent immunomodulator that has been associated with the clinical development of autoimmune disorders. However, diabetes mellitus has not been reported in patients treated with single-agent IL-2. We conducted a clinical trial of a protracted daily schedule of subcutaneously administered low-dose IL-2. A patient with advanced colorectal cancer, treated with 1.5×10⁶ international units of IL-2 daily, developed insulin-requiring diabetes during therapy. Hyperglycemia improved during treatment interruption and recurred with reinstitution of IL-2. The diabetes in this patient developed in the context of T cell and natural killer cell expansion, and the presence of islet cell autoantibodies was documented. We postulate that, in this patient, IL-2 reversed the anergy of autoreactive T cells that had escaped clonal deletion. It is possible that prolonged daily exposure to immunomodulatory doses of IL-2 will result in the development of autoimmune phenomena not observed with other schedules of administration. Received: 15 April 1996 / Accepted: 1 August 1996     

Investigation of the Plasmonic Interaction of Gold Nanoparticles Toward Plasmonic Photothermal Therapeutics

Plasmonic interaction of nanoparticles located in close proximity, embedded in breast tissue, is simulated for estimating the optical characteristics like optical absorption cross-section, plasmonic wavelength as well as full-width half maxima (FWHM). The computations are done for the monomers, homodimers, and heterodimers of spherical and rod-shaped gold nanoparticles considering various interparticle spacings for gold nanospheres and the interparticle spacing as well as the orientation for gold nanorods (GNRs). The results indicate that for the spherical dimer, with the change in interparticle spacing from 1 to 20 nm, the peak absorption cross-section decreases by 43%. Whereas for the GNRs, the absorption cross-section increases/decreases, within 9–18%, depending on the homodimer or heterodimer configuration. Furthermore, secondary peaks for the absorption cross-section are obtained within wavelengths of 630–940 nm due to antibonding modes for GNR heterodimers. For GNR heterodimer located end-to-end, this secondary peak for the absorption cross-section appears at 780 nm irrespective of interparticle spacing within 1–5 nm. The absorption coefficient is considerably dependent on the configuration and proximity of GNRs located within the tissue. While FWHM is not significantly influenced by GNRs configuration and interparticle spacing. For interparticle spacing from 1 to 20 nm, the plasmonic wavelength shifts by 38 nm for the spherical dimer and by 35–86 nm for various GNR dimers. The findings of this study are useful for plasmonic photothermal therapeutics as the heat generation is governed by the resulting absorption cross-section due to plasmonic coupling of the closely spaced and different orientations of the nanoparticles.

     

Trace-Level Detection of Explosive Molecules with Triangular Silver Nanoplates-Based SERS Substrates

We report a simple route to design highly sensitive triangular silver nanoplates (TSNPs)-based SERS substrate for the trace-level detection of explosive molecules. The size-dependent localized surface plasmon resonance (LSPR) tunability for the synthesis of TSNPs is achieved by controlling reaction kinetics and seed volume in a modified seed-mediated approach. The computed extinction spectra of TSNP, using the finite-difference time-domain (FDTD) method, are in excellent agreement with the experimental results, therefore assisting further in the investigation of the plasmonic properties of TSNP. The higher electric field enhancement offered by TSNP is systematically investigated by performing the FDTD simulations for various sizes and corner rounding of TSNP. The FDTD results show that the dipolar plasmon resonance wavelength, size, and corner rounding of TSNP are the principal contributing factors for designing the high-performance SERS substrate. Herein, we have used a portable Raman system for the SERS-based detection of three important explosive molecules: picric acid (PA), ammonium nitrate (AN), and 2, 4-dinitrotoluene (DNT). The TSNP-based SERS substrates display excellent intensity enhancement factors of?~?10⁷ for rhodamine 6G (R6G) and PA and?~?10⁵ for AN. The high sensitivity of SERS substrate with limit-of-detection (LOD) of value 2.3?×?10^?11 M for PA and 3.1?×?10^?8 M for AN and effective batch-to-batch reproducibility for DNT, thus offering its potentials for field detection of explosive molecules at trace-level.

     

Iloprost-induced desensitization of the prostacyclin receptor in isolated rabbit lungs

Background

The rapid desensitization of the human prostacyclin (IP) in response to agonist binding has been shown in cell culture. Phosphorylation of the IP receptor by protein kinase C (PKC) has been suggested to be involved in this process.

Methods and results

In this study we investigated the vasodilatory effects of iloprost, a stable prostacyclin analogue, in perfused rabbit lungs. Continuous infusion of the thromboxane mimetic U46619 was employed to establish stable pulmonary hypertension. A complete loss of the vasodilatory response to iloprost was observed in experiments with continuous iloprost perfusion, maintaining the intravascular concentration of this prostanoid over a 180 min period. When lungs under chronic iloprost infusion were acutely challenged with inhaled iloprost, a corresponding complete loss of vasoreactivity was observed. This desensitization was not dependent on upregulation of cAMP-specific phosphodiesterases or changes in adenylate cyclase activity, as suggested by unaltered dose-response curves to agents directly affecting these enzymes. Application of a prostaglandin E1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) or the PKC inhibitor bisindolylmaleimide I (BIM) enhanced the vasodilatory response to infused iloprost and partially prevented tachyphylaxis.

Conclusion

A three-hour infusion of iloprost in pulmonary hypertensive rabbit lungs results in complete loss of the lung vasodilatory response to this prostanoid. This rapid desensitization is apparently not linked to changes in adenylate cyclase and phosphodiesterase activation, but may involve PKC function and co-stimulation of the EP1 receptor in addition to the IP receptor by this prostacyclin analogue.     

Genome Sequence of a Novel Actinophage PIS136 Isolated from a Strain of Saccharomonospora sp

A wide-host-range bacteriophage (phage) PIS136 was isolated from PA136, a strain of Saccharomonospora belonging to the group actinomycetes. Here, we present the genome sequence of the PIS136 phage, which is 94,870 bp long and contains 132 putative coding sequences and one tRNA gene. An IS element-like region with two genes for putative transposases was identified in the genome. The presence of IS element-like sequences suggests that PIS136 is still under active evolution.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

Consequences of defective tubulin folding on heterodimer levels, mitosis and spindle morphology in Saccharomyces cerevisiae

In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.     

Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP

Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a DeltamxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.