Emission and long-range transport of gaseous mercury from a large-scale Canadian boreal forest fire.

Field observations made at Harvard Forest [Petersham, MA, U.S.A. (42 degrees 54' N, 72 degrees 18' W)] during early July 2002 show clear evidence of long-range transport of gaseous mercury (Hg) in a smoke plume from a series of boreal forest fires in northern Quebec. These measurements indicated significant and highly correlated increases in Hg and CO during the plume event. The Hg:CO emissions ratio determined from the data (8.61 x 10(-8) mol mol(-1)) was combined with previously published information on CO emissions and biomass burned to determine a mean area-based Hg emission flux density for boreal forest fires (1.5 g Hg ha(-1)), annual Hg emissions from Canadian forest fires (3.5 tonnes), and annual global Hg emissions from boreal forest fires (22.5 tonnes). Annual Hg emissions from boreal fires in Canada may equal 30% of annual Canadian anthropogenic emissions in an average fire year and could be as high as 100% during years of intense burning. The Hg:CO emissions ratio of this study was much lower than those reported for a temperate forest in Ontario and a pine/shrub vegetation in South Africa, suggesting that fire emission is dependent on biome/species and that any extrapolation from a single fire event to determine the global fire emission is speculative.

Antitumoral effect of a nonviral interleukin-2 gene therapy is enhanced by combination with 5-fluorouracil.

Using a novel cationic lipid delivery system consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and cholesterol, we delivered murine interleukin-2 (IL-2) cDNA directly into an established murine renal cell carcinoma (Renca). Production of IL-2 within the tumor induced rejection of established tumors (62% on average), whereas control plasmid had little or no effect (17% on average). Surviving animals treated with IL-2:lipid were highly resistant to Renca rechallenge, but not to cross-challenge with a syngeneic mammary adenocarcinoma. Experiments on selectively immunosuppressed animals indicated a requirement for CD8+ T, natural killer, and polymorphonuclear cells. By contrast, depletion of CD4+ T cells did not disrupt the ability of IL-2:lipid to induce tumor rejection. A combination of IL-2 gene therapy with 5-fluorouracil treatment increased the antitumoral efficacy and survival of mice bearing primary and metastatic Renca tumors (42% survival with IL-2:lipid compared with 94% survival with IL-2:lipid plus 5-fluorouracil). These data indicate that rejection of primary and metastatic tumors can be achieved after intratumoral delivery of a nonviral IL-2 gene therapy, and is increased in combination with systemic delivery of a conventional chemotherapeutic agent.

Future molecular approaches to the diagnosis and treatment of glomerular disease.

Current diagnoses and treatment decisions for renal disease are made based upon a combination of clinical and pathological determinations. With the advances in both biochemical and molecular biological techniques, identifying the underlying biochemical and genetic changes that may have initiated and/or contributed to the disease is possible. We describe here technologies that may lead to significant changes in renal disease diagnosis, characterization, treatment, and potentially prevention. For example, differential display techniques and DNA gene chip arrays show the changes in mRNA expression patterns and can potentially identify previously unknown genes and reveal new roles for previously known genes in renal disease. The generation of the single nucleotide polymorphisms (SNP) genomic map will facilitate genetic screening that may identify a gene or combination of genes that produce enhanced disease susceptibility. Combining genomic analysis with epidemiological studies may identify environmental factors that contribute to renal disease onset in genetically susceptible individuals. A number of novel therapies are already on the horizon. These include reagents that abrogate the function of specific cytokines, chemokines, and effector cells. With the list of renal disease genes in hand, their role in renal physiology and pathophysiology can be determined, which should lead to the discovery of pharmacological intervention directed at those genes and their products that play a role in the pathogenesis of renal disease.

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Substrate-specific proteases (BLT-esterase) are localized predominantly in the natural killer cells of unprimed mice.

In leukocytes isolated from unprimed mice, the levels of extractable N alpha-Cbz-Lys-thiobenzylesteresterase (BLT-esterase) closely correlated with the number of natural killer (NK) cells. The spleens of mice that exhibit severe combined immunodeficiency (SCID) contained much higher levels of this enzyme than other mouse strains. Treatments that resulted in a local accumulation of NK cells (as assessed by lytic activity) produced a concomitant increase in BLT-esterase activity. However, short-term in vitro treatment of spleen cells with interferon (IFN)-alpha/beta indicated that BLT-esterase levels correlated more closely with absolute numbers of NK cells than with their lytic capacity. There was a very good correlation between the numbers of cells bearing the NK phenotype (NK-1.1+) and BLT-esterase levels. Cells positively sorted using the NK-specific antibodies NK-1.1 and LGL-1 had high enzymatic activity. The BLT-esterase levels were high in both the NK-1.1+/LGL-1- and NK-1.1+/LGL-1+ subsets. Highly purified CD4+ and CD8+ T cells and sIg+ B cells demonstrated negligible enzyme, as did populations of cells highly enriched for macrophages or neutrophils. However, it should be stressed that the inbred mice used on this study have been maintained in a pathogen-free facility. It would be anticipated that mice maintained under less stringent conditions could exhibit appreciable levels of BLT-esterase activity in their T cells. Nonetheless, BLT-esterase is present at high levels in NK cells and cannot be regarded as a T cell-specific enzyme.

Detachment and lysis of adherent target cells by CD4+ T cell clones involve multiple effector mechanisms.

Detachment of adherent targets from their substratum is a unique activity of both CD8+ and CD4+ T cells. Recently, we have demonstrated that with mixed, alloreactive populations, CD4+ T-cell-mediated detachment is kinetically distinct from lysis and requires protein synthesis. Heterogeneity of effector phenotypes precluded further elucidation of the mechanism of detachment at the mixed population level. Here, we examine further the mechanism of target cell detachment by CD4+ cells using clones which differ in lytic efficiency and demonstrate that detachment of adherent targets from their substratum (1) may result from either protein synthesis-dependent or independent pathways, which can be correlated with differences in clonal lytic phenotype, (2) is contact dependent and does not involve soluble factors, (3) can be pharmacologically uncoupled from IL4 production, as a measure of cytokine release, and (4) fails to correlate with perforin expression by immunocytochemistry. Finally, isolated granule preparations are unable to mediate detachment independent from lysis.

Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

Molecular characterization of the interleukin-8 receptor.

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.

Direct evidence for an intracellular role for tumor necrosis factor-alpha 1. Microinjection of tumor necrosis factor kills target cells.

TNF-alpha is a small peptide cytokine produced primarily by activated macrophages. One of the many biologic activities of TNF is the killing of diverse types of tumor cells. We considered the possibility that killing was mediated by TNF itself at an intracellular site, subsequent to receptor-mediated endocytosis. To test this hypothesis, we microinjected TNF into various murine normal cells and cell lines, some of which were killed by TNF given by the usual extracellular route, and others that were not. Cytotoxic effects of microinjected TNF were observed in several cell types 2 to 4 h after injection. L929 fibroblasts were killed by either extracellular or intracellular TNF. A TNF-resistant subline of L929 was insensitive to either extracellular or intracellular TNF. L6 fibroblasts were found to be resistant to high doses of TNF given either extracellularly or microinjected. Normal macrophages and the J774 macrophage-like cell line were not killed by extracellular TNF, but were rapidly killed by microinjected TNF. Thus, TNF, an extracellular peptide ligand, has an intracellular activity, suggesting that internalization of this ligand may have important intracellular biochemical roles.

The toxicity of rat large granular lymphocyte tumor cells and their cytoplasmic granules for rodent and African trypanosomes.

To explain previous findings that rodent and African trypanosomes are relatively insusceptible to the actions of NK cells, their sensitivity to the cytotoxicity of rat LGL tumor cells and isolated cytolysin-containing granules was studied. LGL tumor cells displayed modest spontaneous killing of rodent trypanosomes but were considerably more effective in the antibody-dependent cell-mediated cytotoxicity mode in the presence of specific antibody. The trypanosomes were quite resistant to lysis by the cytolysin-containing granules, compared with other types of cells. The slow inefficient lysis that occurred in the presence of divalent cations involved granule concentrations thousands of times greater than was required for lysis of SRBC. Rodent trypanosomes were significantly more susceptible to lysis when divested of their surface glycoprotein coats. In the absence (or near absence) of divalent cations, a substance (or substances) present in the granules rapidly destroyed intact and nude trypanosomes; this activity probably was not associated with cytolysin. The rate and efficiency with which the divalent cation-independent substance destroyed three species of trypanosomes indicate that this material deserves further study with an eye to its potential use as a therapeutic agent.

Possible involvement of CTL granule proteases in target cell DNA breakdown.

We have carried out experiments to test whether the granule exocytosis model for lymphocyte cytotoxicity can account for the rapid target DNA breakdown seen during CTL-induced cytotoxicity. Dense granules isolated from cloned mouse CTL and from rat NK tumor cells cause target DNA breakdown during granule cytolysin-mediated lysis of tumor cells, while the purified granule cytolysin caused lysis without DNA breakdown. When target cells are permeabilized with detergent, granule extracts have the ability to release 125I-DNA from nuclei in the absence of detectable cytolysin activity. This activity formed the basis for a nuclear DNA release (NDR) assay; this activity was a property of dense granules of cytotoxic lymphocytes but generally not of other types of lymphoid cells. NDR activity in NK tumor granules had a pH optimum of 7 and was inhibited by micromolar levels of Zn+2, and could be purified away from the granule cytolysin by column chromatography. NDR activity in CTL dense granules could be inactivated by submillimolar concentrations of the protease inhibitors PMSF and DFP (but not soybean trypsin inhibitor or TLCK). In support of the relevance to CTL cytotoxicity of these findings with the NDR assay, pretreatment of CTL with PMSF in the presence of agents raising the intragranular pH inactivated 125I-DNA release from target cells (but not the 51Cr release). These results suggest that a CTL granule component(s), probably a protease, is required for target DNA breakdown.

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.

The tumor disappearance reaction: an in vivo effect of a noncytotoxic lymphokine active against tumor cells.

A lymphokine, tumor migration inhibition factor (TMIF), that can inhibit the migration of tumor cells in vitro without cytotoxic effect has been described. TMIF can be produced in vitro or in vivo. In the present study, it is demonstrated that administration of TMIF can lead to a transient decrease in recoverable tumor cells from the peritoneal cavities of otherwise untreated mice. This result, which appears to be due to a direct effect of the mediator, rather than a consequence of inflammatory cell accumulation, is analogous to the effect of macrophage migration inhibition factor (MIF) on macrophages in the well known macrophage disappearance reaction. These findings demonstrate that TMIF can exert an effect in vivo predicted from its in vitro properties, and raise the possibility that this effect can be exploited in an appropriate therapeutic model.

Effect of phorbol esters on alloimmune cytolysis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) profoundly affects cytolytic T-lymphocyte activity. Alloimmune C57BL/6 (H-2b anti-H-2d) cytolytic splenocytes treated with TPA, 0.3 to 3.0 ng/ml, killed specific P815 (H-2d) targets significantly better than did untreated controls as measured in 4-hr 51Cr release microcytotoxicity assay. Higher concentrations of TPA in the 30- to 100-ng/ml range significantly inhibited cytolytic function. The non-tumor-promoting analog, 4 alpha-phorbol-12,13-didecanoate, failed to affect killing at all doses tested. TPA-induced augmentation of cytolytic function requires an immunologically sensitized splenocyte population, since normal nonimmunized splenocytes treated with TPA did not kill target cells. Furthermore, treatment of splenocytes with anti-Thy 1,2 antibody and complement abrogated killing, indicating that T-lymphocytes mediate the killing. The TPA-induced effect does not require macrophage-like cells, since augmented killing occurred despite the removal of glass-adherent or iron-phagocytosing cells. Finally, the cytolytically augmented effector cells remain immunologically specific, since the nonspecific targets, syngeneic EL4 (H-2b) and third-party L929 (H-2k), are not killed. Thus, low levels of TPA augment the cytolytic ability of alloimmune T-lymphocytes against their specific target cells.

Comparative toxicities of halothane, isoflurane, and diethyl ether at subanesthetic concentrations in laboratory animals.

Effects of 35-day exposures to subanesthetic concentrations of halothane, isoflurane, and diethyl ether were measured in mice, rats, and guinea pigs which were in a phase of rapid body growth. Halothane produced a greater decrement in weight gain and a greater incidence of hepatic degenerative changes than isoflurane or diethyl ether despite its administration at lower anesthetic concentrations. Isoflurane results were intermediate between those of halothane and diethyl ether. No consistent injury to any organ other than the liver was found.