Regional differences in thermoregulation between two European butterfly communities.

Understanding how different organisms cope with changing temperatures is vital for predicting future species' distributions and highlighting those at risk from climate change. As ectotherms, butterflies are sensitive to temperature changes, but the factors affecting butterfly thermoregulation are not fully understood. We investigated which factors influence thermoregulatory ability in a subset of the Mediterranean butterfly community. We measured adult thoracic temperature and environmental temperature (787 butterflies; 23 species) and compared buffering ability (defined as the ability to maintain a consistent body temperature across a range of air temperatures) and buffering mechanisms to previously published results from Great Britain. Finally, we tested whether thermoregulatory ability could explain species' demographic trends in Catalonia. The sampled sites in each region differ climatically, with higher temperatures and solar radiation but lower wind speeds in the Catalan sites. Both butterfly communities show nonlinear responses to temperature, suggesting a change in behaviour from heat-seeking to heat avoidance at approximately 22°C. However, the communities differ in the use of buffering mechanisms, with British populations depending more on microclimates for thermoregulation compared to Catalan populations. Contrary to the results from British populations, we did not find a relationship between region-wide demographic trends and butterfly thermoregulation, which may be due to the interplay between thermoregulation and the habitat changes occurring in each region. Thus, although Catalan butterfly populations seem to be able to thermoregulate successfully at present, evidence of heat avoidance suggests this situation may change in the future.

Consistent oviposition preferences of the Duke of Burgundy butterfly over 14 years on a chalk grassland reserve in Bedfordshire, UK.

Abstract: The Duke of Burgundy butterfly (Hamearis lucina) is known to have specific habitat requirements for its larval foodplants. However, no studies have yet investigated whether these preferences vary over time or in relation to climate, and there is a paucity of data on whether management on reserves can replicate preferred conditions. Here, we build upon existing research to confirm which characteristics Duke of Burgundy prefer for their larval foodplants, whether preferences remain consistent across years, and whether conservation management on reserves can replicate these conditions. Fieldwork was carried out at Totternhoe Quarry Reserve, a chalk grassland site in Bedfordshire, UK. Confirming previous research, we found that large Primula plants in dense patches were chosen for oviposition, but that once chosen there was no preference to lay eggs on a plant's largest leaf. Chosen foodplants were also more sheltered and in closer proximity to scrub than their controls. However, at a finer scale, we found little evidence for any preference based on differences in microclimate, or vegetation height immediately surrounding the plants. This suggests features that alter microclimatic conditions at a larger scale are relatively more important for determining the suitability of oviposition sites. Nearly all preferences remained consistent over time and did not vary between years. Management of scrub on the reserve was able to reproduce some preferred habitat features (high plant density), but not others (large plant size). Implications for insect conservation: The consistency of findings across years, despite inter-annual variation in temperature, rainfall and number of adults, indicates that the Duke of Burgundy is conservative in its foodplant choice, highlighting its need for specific habitat management. Targeted management for foodplants could form part of a tractable set of tools to support Duke of Burgundy numbers on reserves, but a careful balance is needed to avoid scrub clearance leaving plants in sub-optimal conditions.

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia.

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.

Statistics of the contrast of coherent images.

Contrast optimization, also known as image sharpening, is a method that can be used to estimate phase errors in coherent images. However, the contrast measure of a coherent image is a random variable because of the speckle present in coherent images. The variance of this measure puts a limit on the ability of contrast optimization to focus an image. We derive the probability distribution function of the most common contrast measure, the sum of the pixel intensities raised to a power. These statistics are then verified by a number of speckle simulations and compared with measured statistics from synthetic aperture sonar images. The developed statistics can be used as a tool to understand and improve the method of contrast optimization as well as assess its performance for a given imaging system. They can also be used to predict the effect of certain image processing operations on the contrast.

Macrophage colony-stimulating factor antagonists inhibit replication of HIV-1 in human macrophages.

Macrophages infected with HIV-1 produce high levels of M-CSF and macrophage-inflammatory protein-1alpha (MIP-1alpha). M-CSF facilitates the growth and differentiation of macrophages, while the chemotactic properties of MIP-1alpha attract both T lymphocytes and macrophages to the site of HIV infection. Studies described in this work indicate M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and data suggest possible therapeutic strategies for decreasing viral load following HIV infection. We show that macrophage infection with measles virus or respiratory syncytial virus, in contrast to HIV-1, results in production of MIP-1alpha, but not M-CSF. Thus, M-CSF appears to be specifically produced upon infection of macrophages with HIV-1. Furthermore, addition of M-CSF antagonists to HIV-1-infected macrophages, including anti-M-CSF monoclonal or polyclonal Abs or soluble M-CSF receptors, dramatically inhibited HIV-1 replication and reduced production of MIP-1alpha. Our results suggest that biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-1 by 1) blocking virus replication in macrophages, 2) reducing recruitment of HIV-susceptible T cells and macrophages by MIP-1alpha, and 3) preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.

Disparate intracellular processing of human IL-12 preprotein subunits: atypical processing of the P35 signal peptide.

IL-12 is a heterodimeric cytokine produced by APC that critically regulates cell-mediated immunity. Because of its crucial function during immune responses, IL-12 production is stringently regulated, in part through transcriptional control of its p35 subunit, which requires the differentiative effects of IFN-gamma for expression. To determine whether post-transcriptional aspects of IL-12 production might be regulated, we examined intracellular protein processing of each subunit. We report here that p40 and p35 subunits are processed by disparate pathways. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with translocation into the endoplasmic reticulum (ER), processing of p35 does not. Translocation of the p35 preprotein into the ER was not accompanied by cleavage of the signal peptide; rather, removal of the p35 signal peptide occurred via two sequential cleavages. The first cleavage took place within the ER, and the cleavage site localized to the middle of the hydrophobic region of the signal peptide. Although the preprotein was glycosylated upon entry into the ER, its glycosylation status did not affect primary cleavage. Subsequently, the remaining portion of the p35 signal peptide was removed by a second cleavage, possibly involving a metalloprotease, concomitant with additional glycosylation and secretion. Secretion could be inhibited by mutation of the second cleavage site or by inhibition of glycosylation with tunicamycin. In contrast, p40 secretion was not affected by inhibition of glycosylation. Our findings demonstrate that IL-12 subunits are processed by disparate pathways and suggest new modalities for regulation of IL-12 production.

Interferon-gamma-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells.

Interleukin-12 (IL-12) production by human monocytes is stringently regulated through the inducibility of both subunits, p35 and p40, and expression of p35 mRNA is the limiting factor for the secretion of the bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires priming of the monocytes by interferon-gamma (IFN-gamma), followed by brief exposure to lipopolysaccharide or other bacterial products. To investigate control of p35 gene expression, we isolated genomic clones containing the human p35 gene and determined the 5' end of the mRNA expressed in monocytes. We discovered that a unique p35 transcript is induced in monocytes that begins downstream of a consensus TATA box that lies within the 5' end of the cDNA originally cloned from Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by Northern blotting showed that the message from monocytes is approximately 200 bases shorter than message derived from the EBV-transformed B-cell line VDS. The initiation sites downstream from the TATA box were confirmed by RNase protection and 5' RACE. The data indicate that p35 transcription can initiate from different sites depending on the cell type and that the shorter inducible transcript in monocytes is the one that accumulates after stimulation. Protein translation of these two forms may result in proteins of different sizes with potential implications for the regulation of IL-12 secretion and function.

Interleukin-2 inhibits HIV-1 replication in human macrophages by modulating expression of CD4 and CC-chemokine receptor-5.

OBJECTIVE: To determine the effect of recombinant human interleukin (IL)-2 on HIV-1 replication and macrophage colony stimulating factor (M-CSF) production by HIV-1-infected monocyte-derived macrophages (MDM). DESIGN: Therapeutic use of IL-2 increases the number and function of CD4+ T cells. IL-2 also increases M-CSF production and M-CSF receptor expression by human monocytes, but the subsequent effects on HIV-1 replication in MDM have yet to be determined. MDM from HIV-1-seronegative donors were cultured in the presence and absence of IL-2 and infected with HIV-1. Harvested supernatants were monitored for reverse transcriptase activity and M-CSF production. RESULTS: Reverse transcriptase activity was significantly lower when MDM cultures were treated with IL-2 for 10 days prior to infection with HIV-1. IL-2 did not stimulate production of inhibitory chemokines or cytokines, but FACS analysis revealed that expression of CD4, the primary HIV-1 receptor, and CC-chemokine receptor-5, a coreceptor used by macrophage-tropic viruses, are down modulated after treatment with IL-2. CONCLUSION: IL-2 may not only be of benefit in restoring immune function in AIDS patients, but may also help to prevent the infection of healthy macrophages by decreasing their expression of HIV-1 receptors.

Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development.

We have investigated the role of type I IFNs (IFN-alpha and -beta) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-alphabeta to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-gamma, TNF-alpha, and LT secretion. Diminished T cell IFN-gamma production correlated with IFN-alphabeta-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-gamma was not due to increased levels of IL-10 in these cultures, and production of IFN-gamma could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-gamma-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-beta, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-alphabeta inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.

STAT5A-deficient mice demonstrate a defect in granulocyte-macrophage colony-stimulating factor-induced proliferation and gene expression.

Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow-derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the beta-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF-dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF-induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes.

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.

Regulation of interleukin-12 expression in human monocytes: selective priming by interferon-gamma of lipopolysaccharide-inducible p35 and p40 genes.

Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that is critical for T lymphocyte and natural killer cell activities and functions. In this study, we examined the regulation of IL-12 expression by human monocytes in response to bacterial lipopolysaccharide (LPS). Several novel aspects of IL-12 induction from monocytes were shown. Optimal expression of IL-12 mRNA and bioactivity required specific priming of monocytes by interferon-gamma (IFN-gamma) before LPS stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) provided an equivalent priming stimulus for LPS-induced tumor necrosis factor (TNF) and IL-12 p40 mRNA, but primed poorly for LPS-inducible p35 message and secreted IL-12 activity. Macrophage colony-stimulating factor (M-CSF), although a potent survival factor for monocytes, showed no priming activity for IL-12 production. Time course experiments demonstrated independent regulation of p40 and p35 by IFN-gamma and LPS. LPS inducibility of p40 expression required only a brief exposure to IFN-gamma (2 hours), while prolonged exposure (+/- 24 hours) to IFN-gamma resulted in diminishing levels of p40 mRNA. p35 inducibility (by LPS) required a longer exposure to IFN-gamma (8 to 16 hours), and continued to be inducible up to 40 hours following IFN-gamma priming. Both mRNAs were rapidly induced (1 to 2 hours) in IFN-gamma-primed monocytes; p35 message reached a plateau by 2 hours, while p40 continued to accumulate. Finally, both p40 and p35 were directly induced by LPS in the presence of cycloheximide. These results indicated that both p40 and p35 are LPS-inducible in monocytes following IFN-gamma pretreatment, and that the regulated expression of p35 controls the level of active IL-12 protein in purified human monocytes. The selectivity of priming by IFN-gamma is in accord with a putative role for IL-12 in the initiation and amplification of TH1-type responses.

IFN-gamma priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability.

The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by interferon gamma (IFN-gamma), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by IFN-gamma was primarily manifested at the level of TNF mRNA accumulation. IFN-gamma pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either IFN-gamma or GM-CSF, but not M-CSF. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in nuclear factor-kappa B activity in these cells as determined by electrophoretic mobility shift assay using a consensus NF-kappa B oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8-fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of the foreign partheonogenetic tick Amblyomma rotundatum (Acari: Ixodidae) in Florida.

The parthenogenetic tick Amblyomma rotundatum, a Central and South American species, has become established in southern Florida. The date of introduction is unknown, but it is suspected to be either during the 1930s, when 1 of its natural hosts, the giant or marine toad, Bufo marinus, was introduced to southern Florida as a potential biological control of pest beetles in sugar cane fields, or between 1955 and 1964 when specimens of B. marinus were accidentally or deliberately released in the greater Miami area. Several museum specimens of this toad collected in the Miami area 25 April 1979 had nymphal and adult A. rotundatum attached. Subsequent examination of living giant toads collected at another Miami area site from 1983 through 1985 revealed larval, nymphal, and adult A. rotundatum and confirmed colonization of this tick. Under laboratory conditions, another neotropical amphibian and reptile tick, Amblyomma dissimile, is capable of transmitting Cowdria ruminantium, the causative agent of heartwater, a disease present in the Caribbean area. Therefore, we suggest that A. rotundatum should also be tested for vectorial competence.

Priming of human monocytes for enhanced lipopolysaccharide responses: expression of alpha interferon, interferon regulatory factors, and tumor necrosis factor.

Culture of human monocytes with either granulocyte-macrophage colony-stimulating factor or gamma interferon (IFN-gamma) results in a primed state, during which these cells express heightened responses to bacterial lipopolysaccharide (LPS). The production of IFN-alpha in response to LPS by human monocytes has an absolute requirement for priming. Tumor necrosis factor (TNF) expression is also greatly enhanced in primed monocytes after LPS stimulation, but unlike IFN-alpha, TNF is readily expressed in unprimed monocytes as well. In an effort to determine the molecular events associated with IFN-alpha induction in this system, freshly isolated human monocytes were primed by culture with either IFN-gamma or granulocyte-macrophage colony-stimulating factor and then treated with LPS; expression of IFN-alpha subtype 2 (IFN-alpha 2), IFN regulatory factors (IRFs), and TNF was assessed by Northern (RNA blot) analysis. IRF-1 mRNA is expressed at high levels in monocytes and is regulated by both LPS and priming cytokines, but its expression alone does not correlate with the induction of IFN-alpha 2 expression. IRF-2 mRNA is expressed in a more gradual manner following LPS stimulation, implying a possible feedback mechanism for inhibiting IFN-alpha expression. However, nuclear run-on analysis indicates that IFN-alpha 2 is not transcriptionally modulated in this system, in striking contrast to TNF, which is clearly regulated at the transcriptional level. In addition, IFN-alpha 2 mRNA accumulation is superinduced when primed monocytes are treated with LPS plus cycloheximide, while TNF mRNA is relatively unaffected. The results demonstrate that priming can affect subsequent LPS-induced gene expression at different levels in human monocytes.

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A.

The rapid breakdown of target cell DNA during CTL-mediated lysis has been difficult to explain by the granule exocytosis model of cytotoxicity. The involvement of CTL granule proteases in this process was strongly suggested by experiments in which CTL were pretreated with the serine protease inhibitor PMSF, in combination with agents that raise the pH of acidic intracellular compartments. While PMSF pretreatment alone had little effect on target lysis or DNA breakdown, the combination of PMSF and NH4Cl or monensin profoundly reduced target cell DNA release, while little effect was observed on target lysis, as measured by 51Cr release. CTL granule extracts cause release of 125I-DNA from detergent-permeabilized cells. This nuclear DNA-releasing (NDR) activity is inhibited by serine esterase inhibitors that also inhibit the granule BLT-esterase activity, and is specifically immunoabsorbed by antibodies to the CTL granule protease granzyme A. The NDR activity comigrates with BLT-esterase activity during subcellular fractionation, solubilization, gel filtration, and aprotinin-Sepharose affinity chromatography. SDS-PAGE analysis of the affinity-purified product indicates a molecular mass of 60,000 daltons under non-reducing conditions, which moves to 30,000 daltons upon reduction, consistent with previously reported behavior of granzyme A. When the purified material was reduced and alkylated, both esterase and NDR activities comigrated at 30,000 daltons upon gel filtration. Although fully lytic concentrations of purified LGL granule cytolysin alone failed to induce target cell DNA release, a combination of purified granzyme A and the cytolysin induces substantial DNA release.

Causal association of interferon-gamma with tumor regression.

Mouse interferon-gamma (MuIFN-gamma) can cause the rejection of malignant cells in vivo. The evidence presented here in support of this claim includes, first, that spontaneous regression of MSC sarcomas was associated with high intratumoral concentrations of endogenously-produced MuIFN-gamma. By contrast, progressively growing, lethal neoplasms of the same kind invariably contained very little IFN-gamma. Second, spontaneously regressing MSC sarcomas were converted into progressively growing, lethal neoplasms by injecting mice with a monoclonal antibody that neutralized the biological effects of endogenous IFN-gamma. Another monoclonal antibody that bound to, but did not neutralize, mouse IFN-gamma had no effect on the course of tumor regression. Together, these data causally relate MuIFN-gamma to the successful rejection of malignant cells in vivo. They also suggest that findings of poor therapeutic efficacy for IFN-gamma are probably attributable to problems other than an intrinsic lack in the biological activity of the lymphokine.

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.