Immunocytochemical identification of metallothionein- positive cells in rheumatoid synovium and analysis of their cell lineage

Metallothionein is a ubiquitous low molecular weight metalloprotein with powerful protective properties against oxygen radical-mediated cytotoxicity associated with inflammatory processes. In rheumatoid arthritis, the inflammatory damage to the synovium appears to be mediated by free radicals released by the high concentration of neutrophils found in the synovial fluid of the inflamed joint. Synovial tissue obtained during routine surgery on rheumatoid and non-rheumatoid joints was subjected to an indirect immunoperoxidase protocol for the immunolocalization of metallothionein using mouse monoclonal anti-metallothionein antibody E9, reactive against the two major isoforms of mammalian metallothionein. A layer of large dendritic-like cells situated subsynovially in the rheumatoid synovium stained very positively for the metalloprotein, both cytoplasmically and in their nuclei. These cells were not found in non-rheumatoid osteoarthritic or in undamaged synovial tissue associated with traumatic joint injury. An attempt was made to investigate their lineage using a series of antibody markers against epithelial cells, endothelial cells, smooth muscle, mesothelial cells, fibroblasts, neutrophils, dermal dendrocytes, macrophages, low and high molecular weight cytokeratin as well as a cell proliferation marker. From our results, it is suggested that these metallothionein-positive cells are probably myofibroblasts similar to the highly motile cells present in granulation tissue. They may originate from perivascular areas of synovium and their movement into the inflamed synovium may reflect the cytoprotective role of metallothionein acting as a free radical scavenger against oxidative damage     

Induction of apoptosis in neoplastic cells by depletion of vitamin B12

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.     

Survival of Xenorhabdus nematophilus and Photorhabdus luminescens in water and soil

Strains of Xenorhabdus nematophilus and Photorhabdus luminescens were genetically marked with kanamycin resistance and the xylE gene to aid theirdetection in water and soil. Following release in river water, cells declined to undetectable levelsin 6 d. In sterile river water, this decline was enhanced with cells detectable for only 2 d. In sterileMilli-Q purified water, the decline was slower than in either sterile or non-sterile river water.Survival in soil was also restricted with cells only detectable for 7 d. These experiments indicatedthat both X. nematophilus and P. luminescens have limited survival orcompetitive abilities in these environments. The faster decline of populations in sterile river waterwas unexpected, and the possible formation of specialized survival stages was investigated. Insterile water, a non-culturable but viable population of cells was detected, indicating that cellsmay survive longer than anticipated in the environment and remain undetectable using standardmicrobiological methods. The implications of this work to the use of these strains in biologicalcontrol and the release of genetically-modified micro-organisms is discussed.     

Seasonal patterns of egg parasitism and natural biological control of rice brown planthopper in Indonesia

1 Egg mortality is known to be an important factor in brown planthopper (BPH) (Nilaparvata lugens) population dynamics in tropical Asia, but few quantitative data are available on the role of egg parasitoids. 2 Field studies are reported at two sites in Java, Indonesia, over a period of 4 years in both wet and dry seasons, where no chemical pesticides were used. Rice plants previously infested in the laboratory with BPH eggs were exposed to natural parasitism and predation in experimental fields for periods of 5 days. 3 Egg batches were dissected from both experimental and control plants after field exposure at seven fortnightly intervals through the rice growing season and BPH nymphs and adult parasitoids were allowed to emerge. 4 Parasitism by species of Oligosita (Hymenopera, Trichogrammatidae) and Anagrus (Hym., Mymaridae) varied between 18% and 61% in dry, and from 1% to 65% in wet, seasons. There was generally a trend from low to higher rates through wet seasons, contrasting with more uniform higher levels through dry seasons. 5 Reduction in BPH survival as a result of egg parasitism varied between 29% and 91%. Generally parasitism was density independent. 6 It is suggested that lower initial levels of parasitism in dry seasons may be due to the longer fallow period between dry and wet, than between wet and dry, resulting in lower local populations of appropriate natural enemies. 7 Unlike previous studies in Asia, little evidence was found for egg predation as an important mortality factor. 8 The conservation of egg parasitoids should form part of any rice pest management programmes in tropical Asia.     

COUNTING STELLER SEA LION PUPS IN ALASKA: AN EVALUATION OF MEDIUMFORMAT, COLOR AERIAL PHOTOGRAPHY

Estimates of Steller sea lion ( Eumetopias jubatus ) pup production are valuable for estimating population trend and size. Currently in Alaska, pups are counted by visiting rookeries, driving older animals into the water, then walking through the rookeries and counting the pups, a highly disruptive procedure. At smaller rookeries, with good vantage points, pups are occasionally counted from the periphery of rookeries without disturbing the sea lions. We evaluated counts made from medium-format, color, aerial photographs as an alternative to drive counts and peripheral counts. Neither the peripheral counts nor the aerial photographic counts disturbed animals on the rokeries. There were strong 1:1 linear relationships between photographic counts and drive counts ( r ²= 0.966, P < 0.001) and between photographic counts and peripheral counts ( r ²= 0.999, P < 0.001). Precision was similar for all three methods of counting. We suggest that medium-format, color, aerial photographs is appropriate for routine surveys of Steller sea lion pups in Alaska because it is not disruptive to the hauled-out sea lions and provides comparable estimates with similar precision to drive and peripheral counts. Large areas canbe rapidly surveyed during periods of good weather with a minimum of manpower.     

Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue.

Alpha 7 nicotinic acetylcholine receptors are involved in learning and memory, and are implicated in the pathology of Alzheimer's disease and schizophrenia. Detection of alpha7 subunits can be accomplished via immunodetection or alpha-bungarotoxin-binding techniques. Standard protocols for immunohistochemistry and Western blotting were followed using several commercially available antibodies. Various mice were evaluated, including non-transgenics, APP, PS1, APP+PS1, and alpha7 knockouts. Initial results with amyloid-depositing mice revealed alpha7 immunolabeled astrocytes, in addition to expected neuronal staining. Subsequent studies with intrahippocampal injections of lipopolysaccharide (LPS) into alpha7 knockout mice showed that both neuronal and astrocytic labeling by alpha7 antibodies was nonspecific. On Western blots of mouse brain proteins, none of the bands detected with antibodies directed against alpha7 subunits diminished in the alpha7 knockout mice. Although LPS-related changes in the expression of some bands were found, these also were unaffected by the alpha7 genotype of the mice. In general, the Western staining patterns for these antibodies revealed few overlapping bands. These immunodetection data are in contrast to genotyping results and mRNA analyses that confirmed the disruption of the alpha7 allele and lack of alpha7 message in the knockouts. These findings suggest caution in interpreting results when using several commercially available alpha7 nicotinic receptor antibodies.     

Role of sensory input from the lungs in control of muscle sympathetic nerve activity during and after apnea in humans.

We reasoned that, if the lung inflation reflex contributes importantly to apnea-induced sympathetic activation, such activation would be attenuated in bilateral lung transplant recipients (LTX). We measured muscle sympathetic nerve activity (MSNA; intraneural electrodes), heart rate, mean arterial pressure, tidal volume, end-tidal Pco(2), and arterial oxygen saturation in seven LTX and seven healthy control subjects (Con) before, during, and after 20-s end-expiratory breath holds. Our evidence for denervation in LTX was 1) greatly attenuated respiratory sinus arrhythmia and 2) absence of cough reflex below the level of the carina. During apnea, the temporal pattern and the peak increase in MSNA were virtually identical in LTX and Con (347 +/- 99 and 359 +/- 46% of baseline, respectively; P > 0.05). In contrast, the amount of MSNA present in the first 5 s after resumption of breathing was greater in LTX vs. Con (101 +/- 4 vs. 38 +/- 7% of baseline, respectively; P < 0.05). There were no between-group differences in apnea-induced hypoxemia or hypercapnia, hemodynamic, or ventilatory responses. Thus cessation of the rhythmic sympathoinhibitory feedback that normally accompanies eupneic breathing does not contribute importantly to sympathetic excitation during apnea. In contrast, vagal afferent input elicited by hyperventilation-induced lung stretch plays an important role in the profound, rapid sympathetic inhibition that occurs after resumption of breathing after apnea.     

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.     

Number of Abeta inoculations in APP+PS1 transgenic mice influences antibody titers, microglial activation, and congophilic plaque levels.

There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimer's disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.