Macrophage function in the elderly and impact on injury repair and cancer.

Older age is associated with deteriorating health, including escalating risk of diseases such as cancer, and a diminished ability to repair following injury. This rise in age-related diseases/co-morbidities is associated with changes to immune function, including in myeloid cells, and is related to immunosenescence. Immunosenescence reflects age-related changes associated with immune dysfunction and is accompanied by low-grade chronic inflammation or inflammageing. This is characterised by increased levels of circulating pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-6. However, in healthy ageing, there is a concomitant age-related escalation in anti-inflammatory cytokines such as transforming growth factor-ß1 (TGF-ß1) and IL-10, which may overcompensate to regulate the pro-inflammatory state. Key inflammatory cells, macrophages, play a role in cancer development and injury repair in young hosts, and we propose that their role in ageing in these scenarios may be more profound. Imbalanced pro- and anti-inflammatory factors during ageing may also have a significant influence on macrophage function and further impact the severity of age-related diseases in which macrophages are known to play a key role. In this brief review we summarise studies describing changes to inflammatory function of macrophages (from various tissues and across sexes) during healthy ageing. We also describe age-related diseases/co-morbidities where macrophages are known to play a key role, focussed on injury repair processes and cancer, plus comment briefly on strategies to correct for these age-related changes.

State-resolved velocity map imaging of surface-scattered molecular flux.

This work describes a novel surface-scattering technique which combines resonance enhanced multiphoton ionization (REMPI) with velocity-map imaging (VMI) to yield quantum-state and 2D velocity component resolved distributions in the scattered molecular flux. As an initial test system, we explore hyperthermal scattering (E(inc) = 21(5) kcal mol(-1)) of jet cooled HCl from Au(111) on atomically flat mica surfaces at 500 K. The resulting images reveal 2D (v(in-plane) and v(out-of-plane)) velocity distributions dominated by two primary features: trapping/thermal-desorption (TD) and a hyperthermal, impulsively scattering (IS) distribution. In particular, the IS component is strongly forward scattered and largely resolved in the velocity map images, which allows us to probe correlations between rotational and translational degrees of freedom in the IS flux without any model dependent deconvolution from the TD fraction. These correlations reveal that HCl molecules which have undergone a large decrease in velocity parallel to scattering plane have actually gained the most rotational energy, reminiscent of a dynamical energy constraint between these two degrees of freedom. The data are reduced to a rotational energy map that correlates with velocity along and normal to the scattering plane, revealing that exchange occurs primarily between rotation and the in-plane kinetic energy component, with v(out-of-plane) playing a relatively minor role.

Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)-negative precursors: differential regulation of Ia expression at different levels of the respiratory tract.

The relative inefficiency of respiratory mucosal immune function during infancy is generally attributed to the immaturity of the neonatal T cell system. However, immune competence in the adult lung has recently been shown to be closely linked to the functional capacity of local networks of intraepithelial dendritic cells (DC). This study examines the density and distribution of these DC throughout the neonatal respiratory tract in rats, focusing particularly on microenvironmental regulation of their class II major histocompatibility complex (MHC) (Ia) expression. In animals housed under dust-controlled conditions, airway epithelial and alveolar Ia+ DC detectable by immunostaining with the monoclonal antibody (mAb) Ox6 are usually not seen until day 2-3 after birth, and adult-equivalent staining patterns are not observed until after weaning. In contrast, the mAb Ox62 detects large numbers of DC in fetal, infant, and adult rat airway epithelium. Costaining of these Ox62+ DC with Ox6 is rare in the neonate and increases progressively throughout infancy, and by weaning Ia+ DC comprised, on average, 65% of the overall intraepithelial DC population. In infant rats, Ia+ DC are observed first at the base of the nasal turbinates, sites of maximum exposure to inhaled particulates, suggesting that their maturation is driven in part by inflammatory stimuli. Consistent with this suggestion, densitometric analysis of Ia staining intensity of individual DC demonstrates that by 2-3 d after birth, Ia expression by nasal epithelial DC was comparable with that of Iahigh epidermal Langerhans cells in adjacent facial skin, at a time when expression by tracheal epithelial DC was 7-10-fold lower. Additionally, the rate of postnatal appearance of Iahigh DC in the airway epithelium was increased by administration of interferon gamma, and decreased by exposure of infant rats to aerosolized steroid. These findings collectively suggest that Ia expression by neonatal respiratory tract DC is locally controlled and can be upregulated by mediators that are produced within the lung and airway epithelium in response to inhalation of proinflammatory stimuli. It was also noted that Ialow neonatal airway DC expressed adult equivalent levels of class I MHC, which suggests differences in capacity to prime for CD8(+)-dependent versus CD4(+)-dependent immunity to inhaled pathogens, during the early postnatal period.

Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli.

A key rate-limiting step in the adaptive immune response at peripheral challenge sites is the transmission of antigen signals to T cells in regional lymph nodes. Recent evidence suggests that specialized dendritic cells (DC) fulfill this surveillance function in the resting state, but their relatively slow turnover in most peripheral tissues brings into question their effectiveness in signaling the arrival of highly pathogenic sources of antigen which require immediate mobilization of the full range of host defenses for maintenance of homeostasis. However, the present report demonstrates that recruitment of a wave of DC into the respiratory tract mucosa is a universal feature of the acute cellular response to local challenge with bacterial, viral, and soluble protein antigens. Consistent with this finding, we also demonstrate that freshly isolated respiratory mucosal DC respond in vitro to a variety of CC chemokines as well as complementary cleavage products and N-formyl-methionyl-leucine-phenylalanine. This suggests that rapid amplification of specific antigen surveillance at peripheral challenge sites is an integral feature of the innate immune response at mucosal surfaces, and serves as an "early warning system" to alert the adaptive immune system to incoming pathogens.

A comparative study of pain experienced during successive mammography examinations in patients with a family history of breast cancer and those who have had breast cancer surgery.

INTRODUCTION: To measure mammography-related pain in two groups of women undergoing regular surveillance as a baseline for future care. METHODS: Following ethical approval, two hundred and forty two women aged 32-84 years (mean 54), were invited by written invitation to participate in the study. Two hundred women accepted the invitation, 100 women had a family history (FH) of breast cancer, 100 had undergone conservative surgery (FU) for breast cancer and were currently asymptomatic. A validated pain scale was used to score the participants' perceived pain before compression based on memory, immediately after compression and one week later. A series of baseline parameters were also captured including compression force, breast size/density, menstrual history and any adverse events following mammography to allow the investigation of relationships. RESULTS: There was a strong correlation (r = 0.79, p < 0.001) between previous pain scores and current pain scores, no significant correlations were found between breast size, breast density or total compression force and pain. Pain scores reduced between previous and current examinations and there was consistency in overall pain scores, despite variations in the compression forces applied. CONCLUSION: Physical side effects from mammography can develop and extend beyond the examination period. Patients' prior experience of pain was the only significant predictor of current pain in this study. IMPLICATIONS FOR PRACTICE: Data on past mammography experiences are essential to improve future pain outcomes. Post-mammography aftercare should be a routine feature of the examination.

Local effector failure in mesothelioma is not mediated by CD4+ CD25+ T-regulator cells.

The aim of the present study was to define the point at which mesothelioma T-cell responses fail in order to design better immunotherapies. A murine model of mesothelioma was used which was established with asbestos. Inoculation of tumour cells into syngeneic mice results in progressing tumours with similar histopathology to human mesothelioma. The tumour cells secrete a marker tumour antigen similar to secreted tumour-associated products, such as mesothelin. The mesothelioma microenvironment contains stromal elements including dendritic cells, effector CD8(+) and CD4(+) T-cells, and CD4(+) T-regulatory (Tregs) cells, all of which are activated in situ, implying chronic inflammation. Tumour antigens are rapidly transported to draining lymph nodes wherein tumour-specific T-cell responses are generated. Despite the generation of potent CD8(+) cytotoxic lymphocyte in lymphoid organs, those that infiltrate tumours cannot restrain tumour growth suggesting local suppression. Splenic Tregs did not suppress protective responses in adoptive transfer experiments suggesting that systemic Tregs play little role in regulating anti-mesothelioma immune responses. Finally, removal of CD25(+) Tregs from the tumour site and lymphoid organs did not alter tumour growth with or without interleukin (IL)-2 or IL-21 immunotherapy. Tregs are not potent regulators of anti-mesothelioma immunity and targeting these cells may not improve results.

Chloride-dependent cation conductance activated during cellular shrinkage.

A chloride (Cl-)-dependent, nonselective cation conductance was activated during cellular shrinkage and inhibited during cellular swelling or by extracellular gadolinium. The shrinking-induced, nonselective cation conductance and the swelling-induced anion conductance appear to function in the regulation of cell volume in airway epithelia. The shrinking-induced cation conductance had an unusual dependence on Cl-: partial replacement of extracellular Cl- with aspartate reduced the magnitude of the shrinking-enhanced current without accompanying changes in the reversal potential. The Cl- dependence of the nonselective cation conductance could provide a mechanism that tightly regulates Cl- secretion and sodium reabsorption in cells under osmotic stress.

High-purity calcium carbonate in freshwater clam shell.

The calcium carbonate in freshwater clam shells is simlilar in purity to that designated reagent grade. A simple reprecipitation of the shell extract results in a product having less Sr and Mg than reagent grade CaCO(3). Clams are harvested commercially, and discarded shells are a high-quality raw material for the production of CaCO(3).

Antitrypanosomal effect of allopurinol: conversion in vivo to aminopyrazolopyrimidine nucleotides by Trypanosoma curzi.

The parasite Trypanosoma cruzi metabolizes allopurinol by a sequential conversion to allopurinol mononucleotide and aminopurinol mononucleotide. The latter is incorporated into RNA. This transformation of a widely used innocuous agent, allopurinol, into a toxic adenine analog appears to account for the antiprotozoan effect of allopurinol. These unique enzymatic activities appear to occur only in T. cruzi and the pathogenic lesihaminae. Allopurinol may serve as a model for the synthesis of similar antiprotozoan agents.

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system.

We isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobase poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

Gene therapy for malignant mesothelioma: beyond the infant years.

Mesothelioma may be particularly well suited for gene therapy treatment owing to its accessibility, allowing both intrapleural and intratumoral gene delivery. At least four gene therapy trials have been carried out in mesothelioma patients, using different vector systems (adenovirus, vaccinia virus, irradiated tumor cells), and different transgenes (herpes simplex virus thymidine kinase (HSVtk) combined with ganciclovir, IL-2, IFN-beta). Although small in scale, these trials have given an inkling of hope for therapeutic efficacy. However, it is clear that gene therapy protocols need to be optimized further. This paper will review progress made in (i) vector development, (ii) defining optimal transgenes, and (iii) gene delivery. Adenoviruses are the most commonly used vectors for gene therapy, and are continuously being improved. With respect to the nature of the transgenes, five categories can be distinguished: (i) 'suicide' or sensitivity genes (e.g., HSVtk), (ii) cytokines and other immune modulators, (iii) replacements for mutant tumor suppressor genes (e.g., p53), (iv) antiangiogenic proteins and (v) tumor antigens. It seems clear that expression of a single transgene is unlikely to be sufficient to eradicate a tumor, such as mesothelioma, that is diagnosed late in disease progression. Hence, multimodality therapy, including conventional therapy (chemo- and radiotherapy, surgery) with one or more transgenes has a higher chance of success.

A streaked X-ray spectroscopy platform for rapidly heated, near-solid density plasmas.

A picosecond, time-resolved, x-ray spectroscopy platform was developed to study the thermal line emission from rapidly heated solid targets containing buried aluminum or iron layers. The targets were driven by high-contrast 1ω or 2ω laser pulses at focused intensities up to 1 × 1019 W/cm2. The experimental platform combines time-integrating and time-resolved x-ray spectrometers. Picosecond time resolution was achieved with a pair of ultrafast x-ray streak cameras coupled to high-throughput Hall spectrometers. Time-integrated spectra were collected on each shot to correct the streaked data for variations in x-ray photocathode spectral sensitivity. The time-integrated spectrometer uses three elliptical crystals to disperse x rays with energies between 800 and 2100 eV with moderate (E/ΔE ∼ 450) resolving power. The streaked spectrometers accept four interchangeable conical crystals with higher resolving power (E/ΔE ∼ 650) to measure the brightest thermal lines in the 1300 to 1700 eV spectral range.

Towards microfluidic reactors for in situ synchrotron infrared studies.

Anodically bonded etched silicon microfluidic devices that allow infrared spectroscopic measurement of solutions are reported. These extend spatially well-resolved in situ infrared measurement to higher temperatures and pressures than previously reported, making them useful for effectively time-resolved measurement of realistic catalytic processes. A data processing technique necessary for the mitigation of interference fringes caused by multiple reflections of the probe beam is also described.

The inwardly rectifying K(+) channel subunit GIRK1 rescues the GIRK2 weaver phenotype.

The weaver (wv) gene has been identified as a glycine to serine substitution at residue 156 in the H5 region of inwardly rectifying K(+) channel, GIRK2. The mutation is permissive for the expression of homotetrameric channels that are nonselective for cations and G-protein-independent. Coexpression of GIRK2wv with GIRK1, GIRK2, or GIRK3 in Xenopus oocytes along with expression of subunit combinations linked as dimers and tetramers was used to investigate the effects of the pore mutation on channel selectivity and gating as a function of relative subunit position and number within a heterotetrameric complex. GIRK1 formed functional, K(+) selective channels with GIRK2 and GIRK3. Coexpression of GIRK2wv with GIRK1 gave rise to a component of K(+)-selective, G-protein-dependent current. Currents resulting from coexpression of GIRK2wv with GIRK2 or GIRK3 were weaver-like. Current from dimers of GIRK1-GIRK2wv, GIRK2-GIRK2wv, and GIRK3-GIRK2wv was phenotypically similar to that obtained from coexpression of monomers. Linked tetramers containing GIRK1 and GIRK2wv in an alternating array gave rise to wild-type, K(+)-selective currents. When two mutant subunits were arranged adjacently in a tetramer, currents were weaver-like. These results support the hypothesis that in specific channel stoichiometries, GIRK1 rescues the weaver phenotype and suggests a basis for the selective neuronal vulnerability that is observed in the weaver mouse.

Functional and biochemical characterization of the human potassium channel Kv1.5 with a transplanted carboxyl-terminal epitope in stable mammalian cell lines.

The role of the C-terminal domain of the hPCN1/Kv1.5 delayed rectifier K+ channel was investigated in transfected stable cell lines employing antipeptide and anti-epitope antibodies against hPCN1-cp, an epitope-fusion gene carrying additional sequences encoding a 32 amino acid C-terminal extension. Both wild-type and chimeric genes showed high levels of K+ channel expression. Detailed electrophysiologic characterization showed there to be no significant effect of the C-terminal extension on channel activity. Immunoblots of whole-cell and membrane preparations demonstrated primarily intact protein in which the C-terminal extension was not cleaved from the peptide chain. Two bands were visualized from cells transfected with either the wild-type or chimeric channels; the slower migrating band was a non-N-glycosylated form. The epitope-fusion method will be a useful adjunct to studying the role of functional domains in ion channels, and may provide a means for rapid affinity purification of channel protein.

Purine metabolism in Leishmania donovani and Leishmania braziliensis.

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Both adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase ((EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors. The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activiting.

Lack of ignorance to tumor antigens: evaluation using nominal antigen transfection and T-cell receptor transgenic lymphocytes in Lyons-Parish analysis--implications for tumor tolerance.

A substantial body of literature has described weak antitumor CTL responses in tumor-bearing hosts, and a number of authors have suggested that tumor tissue in some way sequesters antigen from the immune system, a failure of the tumor-specific immune response largely attributable to "ignorance." To evaluate this in a tumor model, we stably transfected murine tumor cell lines with genes coding for the nominal antigens influenza hemagglutinin (HA) or ovalbumin (OVA) and adoptively transferred HA- or OVA-specific T-cell receptor-transgenic, CD8-positive T cells into mice-bearing these tumors. Tumor antigen cross-presentation within draining lymph nodes (LNs) was then examined using Lyons-Parish analysis, detection of a proliferative response of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled CD8 T cells from T-cell receptor mice using flow cytometric analysis. Our studies demonstrate clearly that tumor antigens are constitutively presented in LNs draining tumors and can stimulate a T-cell proliferative response. This lack of ignorance was not simply attributable to the model chosen, because it was seen with three different cell lines, two different antigens, and two different mouse strains. Furthermore, it occurred regardless of whether these tumor antigens were expressed as cytoplasmic, transmembrane, or secreted proteins. When tumor antigens were present in low concentrations, antigen cross-presentation was not absent but simply delayed. Interestingly, tumor antigen cross-presentation remained localized to the LNs draining the tumor throughout the period of tumor growth. Curiously, in animals where tumors failed to grow, evidence of continued cross-presentation of the tumor antigen was seen up to 6 months after tumor inoculation. These data suggest that ignorance is not an explanation for the failure of the host immune system to respond to tumor antigens.