[Periodic fever and pancytopenia in a 35-year-old patient]. / Periodisches Fieber und Panzytopenie bei einem 35­jährigen Patienten.

MEDICAL HISTORY AND INITIAL PRESENTATION: A 35-year-old patient with a previous history of persistent episodic fever, sore throat, myalgia, and cephalgia presented for evaluation of pancytopenia. He had no recent travel history, except for a stay in Italy 1 year prior to admission and in Spain several years in the past. DIAGNOSTIC WORKUP: Laboratory evaluation confirmed pancytopenia, agranulocytosis, and elevated infection parameters without indicative serological results en par with lymphadenitis colli. Computed tomography scanning revealed cervical lymphadenopathy, hepatosplenomegaly, and colitis with occult perforation of the sigmoid colon. Bone marrow biopsy showed an infiltration of polyclonal plasma cells. Lymph node biopsy was compatible with necrotizing lymphadenitis. DIAGNOSIS: Polymerase chain reaction analysis of a lymph node specimen confirmed the presence of Leishmania species, thereby enabling the diagnosis of visceral Leishmania. THERAPY COURSE: Treatment with liposomal amphotericin B was initiated. Both fever and lymphadenopathy quickly resolved. CONCLUSION: VL is a clinically pleiotropic, severe disease with fatal outcome if left untreated. It often presents with distinct similarities to hematologic malignancies. Exacerbation can occasionally occur as fulminant macrophage activation syndrome. Disease incidence is globally increasing and has not peaked as yet. A complex interplay between pathogen and the immune system is the key pathophysiological mechanism.

Laser-assisted atom probe tomography of semiconductors: The impact of the focused-ion beam specimen preparation.

This paper demonstrates the increased light absorption efficiency of semiconducting atom probe tips resulting from focused-ion-beam (FIB) preparation. We use transmission electron microscopy to show that semiconducting tips prepared with FIB are surrounded with an amorphized shell. Photomodulated optical reflectance measurements then provide evidence that FIB-induced damage leads to an increase in both sub- and supra-bandgap light absorption efficiency. Using laser-assisted atom probe tomography (La-APT) measurements, we finally show that, for a nanoscale tip geometry, the laser-induced heating of a tip during La-APT is enhanced by the FIB preparation. We conclude that, upon supra-bandgap illumination, the presence of a FIB-amorphized surface dramatically increases the light-induced heat generation inside semiconducting tips during La-APT. Furthermore, we also deduce that, in the intriguing case of sub-bandgap illumination, the amorphization plays a crucial role in the unexpected light absorption.

Wet-chemical etching of atom probe tips for artefact free analyses of nanoscaled semiconductor structures.

We introduce an innovative specimen preparation method employing the selectivity of a wet-chemical etching step to improve data quality and success rates in the atom probe analysis of contemporary semiconductor devices. Firstly, on the example of an SiGe fin embedded in SiO2 we demonstrate how the selective removal of SiO2 from the final APT specimen significantly improves accuracy and reliability of the reconstructed data. With the oxide removal, we eliminate the origin of shape artefacts, i.e. the formation of a non-hemispherical tip shape, that are typically observed in the reconstructed volume of complex systems. Secondly, using the same approach, we increase success rates to ∼90% for the damage-free, 3D site-specific localization of short (250 nm), vertical Si nanowires at the specimen apex. The impact of the abrupt emitter radius change that is introduced by this specimen preparation method is evaluated as being minor using field evaporation simulation and comparison of different reconstruction schemes. The Ge content within the SiGe fin as well as the 3D boron distribution in the Si NW as resolved by atom probe analysis are in good agreement with TEM/EDS and ToF-SIMS analysis, respectively.

Atom probe tomography analysis of SiGe fins embedded in SiO2: Facts and artefacts.

We present atom probe analysis of 40nm wide SiGe fins embedded in SiO2 and discuss the root cause of artefacts observed in the reconstructed data. Additionally, we propose a simple data treatment routine, relying on complementary transmission electron microscopy analysis, to improve compositional analysis of the embedded SiGe fins. Using field evaporation simulations, we show that for high oxide to fin width ratios the difference in evaporation field thresholds between SiGe and SiO2 results in a non-hemispherical emitter shape with a negative curvature in the direction across, but not along the fin. This peculiar emitter shape leads to severe local variations in radius and hence in magnification across the emitter apex causing ion trajectory aberrations and crossings. As shown by our experiments and simulations, this translates into unrealistic variations in the detected atom densities and faulty dimensions in the reconstructed volume, with the width of the fin being up to six-fold compressed. Rectification of the faulty dimensions and density variations in the SiGe fin was demonstrated with our dedicated data treatment routine.

Effect of hyperthermia on the antiproliferative activities of murine alpha-, beta-, and gamma-interferon: differential enhancement of murine gamma-interferon.

Fever is frequently an important side effect of interferon (IFN) therapy. Studies have shown that culturing interferon-treated cells at elevated temperature heightens the antiproliferative activity of IFN-alpha and IFN-beta. Since IFN-gamma has also been shown to be a potent antiproliferative agent, the effect of elevated temperature on IFN-gamma activity was compared to its effect on IFN-alpha and IFN-beta. Mouse B-16 melanoma cells were simultaneously cultured under cloning conditions at a range of temperatures (37.3, 38.1, 38.6, and 39.4 degrees C) in the presence of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma. The antiproliferative activities of all three interferons were enhanced by incubation at the elevated temperatures. However, the elevated temperatures had a more dramatic enhancing effect on the antiproliferative activity of MuIFN-gamma (10-fold enhancement) than of either MuIFN-alpha or MuIFN-beta (2.9- and 3.4-fold enhancement, respectively). Next, the enhancing effect of elevated temperature (39.4 degrees C) was examined for a range of interferon concentrations. The degree of the enhancing effect increased with increasing concentrations of MuIFN-gamma but not with increasing concentrations of MuIFN-alpha or MuIFN-beta. Enhancing effects of temperature as high as 14-fold were observed for 100 units of MuIFN-gamma/ml. This dramatic enhancement was observed for both natural and recombinant MuIFN-gamma and was neither a function of greater relative perception of MuIFN-gamma titer at elevated temperature nor a function of greater relative stability of MuIFN-gamma at the elevated temperature. The differential enhancement of MuIFN-gamma activity by elevated temperature appeared to be specific for the antiproliferative activity, since the antiviral activity of MuIFN-gamma was not relatively more enhanced at 39.4 degrees C than were the antiviral activities of MuIFN-alpha and MuIFN-beta. These results suggest that fever may be an important factor in maximizing the antitumor effects of MuIFN-gamma and perhaps of human IFN-gamma. They also raise the possibility that a combination treatment regimen of hyperthermia and interferon therapy, particularly IFN-gamma therapy, may provide a significant antitumor effect.

Effect of hyperthermia on combination interferon treatment: enhancement of the antiproliferative activity against murine B-16 melanoma.

Previous studies have evaluated the effects of hyperthermia on the antiproliferative activity of interferon. The activities of all three types of interferon have been shown to be synergistically enhanced by hyperthermic conditions. Further, the antiproliferative activity of interferon has been shown to be synergistically enhanced by combinations of gamma-plus alpha- or beta-interferon. The question remained whether combining these two methods of enhancing interferon activity would lead to an even higher level of enhancement of antiproliferative activity or to an antagonism of their separate effects. To address this question, mouse B-16 melanoma cells were cloned at 37.3 degrees C and at 39.4 degrees C in the presence of various combinations of murine alpha/beta- and gamma-interferon. Potentiation of interferon's antiproliferative activity by combination interferon treatment was found to occur at both temperatures. Moreover, the level of potentiation was synergistically enhanced by hyperthermic conditions. The results suggest that a combined treatment regimen of hyperthermia and combination interferon therapy (gamma- plus alpha- or beta-interferon) may provide a highly potent antitumor effect.

Insights into the nanoscale lateral and vertical phase separation in organic bulk heterojunctions via scanning probe microscopy.

Solution processed polymer (donor) and fullerene (acceptor) bulk heterojunctions are widely used as the photo active layer in organic solar cells. Intimate mixing of these two materials is essential for efficient charge separation and transport. Identifying relative positions of acceptor and donor rich regions in the bulk heterojunction with nanometer scale precision is crucial in understanding intricate details of operation. In this work, a combination of Ar(+)2000 gas cluster ion beam and scanning probe microscopy is used to examine the lateral and vertical phase separation within regio-regular poly(3-hexylthiophene)(P3HT):phenyl-C60-butyric acid methyl ester (PCBM) bulk heterojunction. While the Ar(+)2000 gas cluster ion beam is used as a sputter tool to expose the underneath layers, scanning probe microscopy techniques are used to obtain two-dimensional (2D) electrical maps (with sub-2 nm lateral resolution). The electrical mapping is decoded to chemical composition, essentially producing lateral and vertical maps of phase separation. Thermal stress causes large PCBM-rich hillocks to form, and consequently affecting the balance of P3HT:PCBM heterojunctions, hence a negative impact on the efficiency of the solar cell. We further developed a method to analyze the efficiency of exciton dissociation based on the current maps and a loss of 20% in efficiency is observed for thermally degraded samples compared to fresh un-annealed samples.

Germline mutations in Dok1 do not predispose to chronic lymphocytic leukemia.

The genetic basis of familial CLL is poorly understood and to date no gene which when mutated in the germline has been unambiguously shown to confer susceptibility to the disease. Dok1 maps to chromosome 2p13, a region commonly rearranged in CLL. Dok1 inhibits MAP kinase activity, down-regulates cell proliferation and has a suppressive effect on cellular transformation and B-cell signalling pathways. A recent report has implicated mutation of Dok1 in the aetiology of CLL. To examine the proposition that germline mutations in Dok1 act as high penetrance susceptibility alleles for CLL we screened 140 familial cases for functional sequence variants. No pathogenic mutations were detected. This result indicates that germline mutations in Dok1 are unlikely to cause an inherited predisposition to CLL.

B16 melanoma cells exposed in vitro to long-term IFN-alpha treatment (B16 alpha cells) as activators of tumor immunity in mice.

Mice inoculated with B16 melanoma cells exposed to long-term in vitro IFN-alpha treatment (> or = 14 days, B16 alpha cells) but not short-term in vitro IFN-alpha treatment (24 h) exhibited an enhanced survival time. Enhanced survival time also occurred when inactivated B16 alpha cells were inoculated at the same time as live B16 cells. Further, an even greater improvement in survival time was observed when the inactivated B16 alpha cells were inoculated before live B16 cell challenge. No enhancement in survival time was observed when mice were inoculated with inactivated, untreated B16 cells. Enhancement of survival time by B16 alpha cells was unrelated to retrovirus surface antigen expression. Long-lasting protective immunity to B16 cells was observed in mice that survived B16 alpha cell, but not normal B16 cell, challenge and subsequent IFN treatment. It is evident that inoculation with inactivated B16 alpha cells, but not with inactivated untreated B16 cells, was able to prolong significantly the survival time of mice either simultaneously or subsequently challenged with live B16 cells. Additionally, survival of B16 alpha-inoculated but not B16-inoculated mice was accompanied by a durable immunity. Inoculation of inactivated B16 alpha cells may serve as a model for the induction of host immunity to a parental primary or secondary tumor.

Enhanced in vivo sensitivity of in vitro interferon-treated B16 melanoma cells to CD8 cells and activated macrophages.

Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-alpha become resistant to the in vitro antiproliferative effects of IFN-alpha. However, IFN-alpha-treated mice inoculated with these in vitro IFN-treated cells (B16 alpha res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-alpha-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16 alpha res cells to the in vivo antitumor effects of IFN-alpha was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16 alpha res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16 alpha res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-alpha against B16 alpha res cells. Macrophage killing was unlikely to be mediated by TNF-alpha or IL-1 as B16 and B16 alpha res cells were equally sensitive to TNF-alpha and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16 alpha res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.

Orally administered IFN-alpha acts alone and in synergistic combination with intraperitoneally administered IFN-gamma to exert an antitumor effect against B16 melanoma in mice.

Administration of interferons (IFN) via the intranasal route recently has been shown to exert an antitumor activity against a variety of tumors in mice, including B16 melanoma inoculated intravenously. This study confirms the antitumor activity of orally administered IFN-alpha against B16 melanoma challenge using another route of tumor inoculation, the intraperitoneal route. It further demonstrates that orally administered IFN-alpha can synergistically interact with intraperitoneally administered IFN-gamma but not with intraperitoneally administered IFN-alpha. The results support the interpretation that the oral route may provide an effective alternative or supplement to current methods of IFN administration for the control of malignancies.

Lack of mda-6/WAF1/CIP1-mediated inhibition of cyclin-dependent kinases in interferon-alpha resistant murine B16 melanoma cells.

Previously we demonstrated that IFN-alpha augments mda-6/WAF1 and inhibits cyclin-dependent kinases in a p53-independent fashion in B 16 murine melanoma cells. On the other hand, IFN-gamma activates p53 expression without affecting the mda-6/WAF1 system. Combination of the two IFNs is additive. B16 cells acquire IFN-alpha resistant but IFN-gamma sensitive phenotype after long term IFN-alpha treatment (B16alpha cells). Here we demonstrate the absence of mda-6/WAF1-associated repression of cyclin-dependent kinases, but the existence of p53-dependent c-myc inhibition in IFN-gamma-treated B16alpha cells. Clearly, selective desensitization of IFN-alpha related growth regulation does not influence the IFN-gamma associated pathway. Our results further support the coexistence of distinct growth regulatory mechanisms in B16 cells that can be activated by different IFN-types independently of each other.

Potentiating effect of murine interferon-gamma-containing lymphokine preparations on the antiviral and antiproliferative effects of murine interferon-alpha/beta: identification of the potentiation factor as murine interferon-gamma itself.

Mixed preparations of murine interferon-gamma (MuIFN-gamma) and murine interferon-alpha/beta (MuIFN-alpha/beta) have been shown to induce more than additive levels of antiviral protection, when compared to those induced by these interferons given separately. MuIFN-gamma preparations contain many lymphokines and several of these as well as MuIFN-gamma itself may participate in this potentiation. In the present study, natural as well as three recombinant DNA-derived MuIFN-gamma's, in combination with antibody to MuIFN-gamma have been employed to examine the precise role of MuIFN-gamma. The antiviral effect was examined with a single cycle virus yield reduction assay and the antiproliferative effect with a colony formation inhibition assay. Recombinant DNA-derived MuIFN-gamma was as effective as natural MuIFN-gamma at participating in the potentiation of both the antiviral and antiproliferative activities. Antibody to MuIFN-gamma effectively blocked the potentiation of both the antiviral and the antiproliferative activities of natural and recombinant DNA-derived MuIFN-gamma's. Since the recombinant DNA-derived preparations from E. colie can be assumed not to contain mammalian proteins other than MuIFN-gamma, the data conclusively demonstrate that the potentiation factor in MuIFN-gamma preparations is MuIFN-gamma itself.

Potentiation of interferon action by mixtures of recombinant DNA-derived human interferons.

Natural and essentially pure recombinant DNA-derived HuIFN-alpha and HuIFN-gamma were examined for their relative abilities to potentiate interferon action. Potentiation of human interferon's antiviral and antiproliferative activities were studied. The essentially pure recombinant DNA-derived human interferons were found to be as effective in their potentiating interactions as their natural counterparts. The results demonstrate that it is the human interferons themselves which interact to potentiate human interferon's varied activities.

Enhancement of MuIFN-gamma antitumor effects by hyperthermia: sequence dependence and time dependence of hyperthermia.

Heat-induced total body hyperthermia has been shown to synergistically enhance the in vivo antitumor effects of MuIFN-gamma directed against B16 melanoma in mice. This observation was made with an 8-hour exposure to hyperthermia that was administered following MuIFN-gamma treatment. It was not known whether this was the most efficacious treatment protocol. Various treatment protocols exploring MuIFN-gamma treatment at different relative times of exposure to hyperthermia and involving different durations of hyperthermia were evaluated. In a 5-day course of therapy, B16 tumor-bearing mice were injected with MuIFN-gamma or mock interferon and incubated in a dry incubator (resulting in a 2 degrees C rise in body temperature). The antitumor efficacy of MuIFN-gamma administered before, in the middle of, or following 8 hours of hyperthermia was evaluated. The antitumor effects of MuIFN-gamma were synergistically enhanced when the MuIFN-gamma was administered before 8 hours of hyperthermia (3.9-fold greater increased life span than with MuIFN-gamma treatment alone). However, when MuIFN-gamma was administered in the middle or following hyperthermia, the life spans were essentially the same as for MuIFN-gamma treatment alone, indicating that the effect of the hyperthermia exposures according to these treatment protocols were less than additive. Administration of MuIFN-gamma before hyperthermia exposures of 2 hours, 5 hours, and 8 hours showed antagonistic, additive, and synergistic interactions of the MuIFN-gamma treatment and hyperthermia exposure, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance to the antiproliferative activity of IFN-alpha: further characterization and demonstration of antagonistic effects of IFN-gamma.

Mouse B16 melanoma cells have been shown to rapidly develop resistance to the antiproliferative effects of MuIFN-alpha or MuIFN-beta when exposed to these interferons. In cloning studies, the maximal antiproliferative effects of MuIFN-alpha were seen with 2-4 days treatment. This resistance has been further characterized. The level of resistance which develops in B16 melanoma cells is dependent upon the concentration of MuIFN-alpha to which the cells are exposed. In addition, B16 melanoma cells which are resistant to the antiproliferative effects of MuIFN-alpha have greatly elevated levels of the interferon-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase. Since it has previously been shown that B16 melanoma cells do not develop resistance to the antiproliferative effects of MuIFN-gamma, several experiments studied the influence of MuIFN-gamma on the development of resistance to MuIFN-alpha. Combinations of IFN-gamma and IFN-alpha have previously been shown to result in a synergistic enhancement of the antiproliferative effects. Kinetic studies show that the response of the cells to the MuIFN-gamma antiproliferative effect appears to be dominant over the development of resistance since no resistance develops in response to combination treatment. Not only is MuIFN-gamma able to prevent development of resistance when it is present continuously, but also when it is used for the sequential treatment of the cells before their exposure to MuIFN-alpha. A 2-day pretreatment with MuIFN-gamma is sufficient to prevent the development of resistance during later exposure of the cells to MuIFN-alpha alone for up to 6 additional days.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta.

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hyperthermia on the in vitro antiproliferative activities of HuIFN-alpha, HuIFN-beta, and rHuIFN-gamma employed separately and in combination.

The relative enhancing effects of hyperthermia on the three types of interferon were evaluated in cloning studies for three human cell lines: G-361 malignant melanoma cells, WISH ammion cells, and AGS stomach adenocarcinoma cells. Hyperthermia enhanced the antiproliferative activity of rHuIFN-gamma against each of the three cell lines and the levels of enhancement by hyperthermia were seen to increase with increasing concentrations of rHuIFN-gamma. The maximum observed levels of enhancement of rHuIFN-gamma activity by hyperthermia varied from cell line to cell line. However, when the relative sensitivities of the cell lines to rHuIFN-gamma were taken into account, the levels of enhancement of rHuIFN-gamma antiproliferative activity by hyperthermia were seen to be similar for each of the cell lines, indicating that hyperthermia consistently enhanced rHuIFN-gamma antiproliferative activity. Hyperthermia did not consistently enhance the antiproliferative activities of HuIFN-alpha and HuIFN-beta. Further studies indicated that hyperthermia enhanced by approximately 6-fold the antiproliferative effects of combinations of rHuIFN-gamma with HuIFN-alpha and HuIFN-beta. The results support the possibility that a combination treatment protocol of hyperthermia and interferon administration (particularly HuIFN-gamma or combinations of HuIFN-gamma with HuIFN-alpha or HuIFN-beta) may provide an enhanced antitumor effect in man.

Enhanced in vitro macrophage cytotoxicity against interferon-treated B16 melanoma cells.

Resistance to the in vitro antiproliferative effects of INF-alpha rapidly develops in mouse B16 melanoma cells that are maintained in vitro in IFN-alpha (B16 alpha res cells). B16 alpha res cells, however, are significantly more sensitive to the antitumor effects of IFN-alpha when they are injected into mice. This enhanced sensitivity appears to be due, at least in part, to activated macrophages. To investigate enhanced macrophage sensitivity of B16 alpha res cells, macrophage-mediated cytotoxicity assays have been performed using both B16 and B16 alpha res cell targets. Thioglycollate-elicited peritoneal macrophages activated in vitro with IFN-gamma exhibited dose-dependent cytotoxicity against both B16 and B16 alpha res cells, but significantly higher levels of cytotoxicity occurred with B16 alpha res targets. Kinetics experiment results showed that the cytolytic effects against B16 alpha res cells occurred at a very much faster rate than the cytolytic effects against B16 cells (50% cytotoxicity with 2 h of incubation versus 40% cytotoxicity by 24 h, respectively). Finally, peritoneal macrophages from B16-inoculated mice also were significantly more cytotoxic against B16 alpha res cells than against B16 cells. Macrophages from B16 alpha res-inoculated mice were significantly more cytotoxic against B16 alpha res cells than were macrophages from B16-inoculated mice. Taken together, these observations provide in vitro evidence to support the suggestion that peritoneal macrophages are important mediators of the enhanced host-mediated antitumor effects against B16 alpha res cells.

Prevention of resistance to IFN-alpha antiproliferative activity: characterization of the effect of IFN-gamma and substitution for IFN-gamma by tumor necrosis factor.

Non-genetic resistance to the antiproliferative effects of interferon-alpha (IFN-alpha) develops in murine and human melanoma cells within 2-4 days of exposure of the cells to IFN-alpha. Simultaneous treatment of murine B16 melanoma cells with MuIFN-gamma and MuIFN-alpha prevents the development of resistance. In this study, the ability of MuIFN-gamma pretreatment to prevent the development of resistance was assessed for varying concentrations of MuIFN-gamma and for varying lengths of time of pretreatment. Pretreatment of the cells for 48 h with MuIFN-gamma using concentrations as low as 5 U/ml prevents the subsequent development of resistance when the cells are cloned in the presence of MuIFN-alpha. Higher concentrations of MuIFN-gamma are more effective in preventing the development of resistance. In addition, short MuIFN-gamma pretreatment times, such as 2-4 h, appeared to be most effective in preventing the development of resistance. In order to determine the mechanism for this biological effect, various second messenger perturbing chemical agents and several other biological agents were screened for ability to prevent the development of resistance. Neither interleukin-2 (IL-2), epidermal growth factor (EGF), nor any of the chemical agents examined could prevent the development of resistance, nor did they alter the ability of MuIFN-gamma to prevent the development of resistance. Tumor necrosis factor (TNF), however, was able to substitute for MuIFN-gamma in preventing the development of resistance, using concentrations of 125 ng/ml and higher.(ABSTRACT TRUNCATED AT 250 WORDS)