Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi's Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell-Specific Manner.

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens and may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action is well understood. We report here that in some cells but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies.

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.

Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1.

IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.

SARS-CoV-2 differs from SARS-CoV in the requirements for receptor expression and proteolytic activation to trigger cell-cell fusion and is not inhibited by Bromhexine

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCECell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease. On several adherent cell types, EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV. KSHV gH/gL also has previously been found to interact weakly with other members of the Eph family of receptor tyrosine kinases (Ephs), and other A-type Ephs have been shown to be able to compensate for the absence of EphA2 using overexpression systems. However, whether these interactions are of functional consequence at endogenous protein levels has remained unclear so far. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells. Knockout of EphA5, the second most expressed A-type Eph in BJAB cells, had a similar, although less pronounced, effect on KSHV infection. Receptor function of EphA7 was conserved for cell-free infection by the related rhesus monkey rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells.IMPORTANCE Infection of B cells is relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman's disease and PEL. Therefore, elucidating the process of B cell infection is important for the understanding of KSHV pathogenesis. While the high-affinity receptor for the gH/gL glycoprotein complex, EphA2, has been shown to function as an entry receptor for various types of adherent cells, the gH/gL complex can also interact with other Eph receptor tyrosine kinases with lower avidity. We analyzed the Eph interactions required for infection of BJAB cells, a model for B cell infection by KSHV. We identified EphA7 as the principal Eph receptor for infection of BJAB cells by KSHV and the related rhesus monkey rhadinovirus. While two analyzed PEL cell lines exhibited high EphA2 and low EphA7 expression, a third PEL cell line, BCBL-1, showed high EphA7 and low EphA2 expression, indicating a possible relevance for KSHV pathology.

Changing life expectancy in central Europe: is there a single reason?

BACKGROUND: During the 1980s, at a time that life expectancy at birth in western Europe has increased by 2.5 years, it has stagnated or, for some groups, declined in the former socialist countries of central and eastern Europe. METHODS: A study was carried out to ascertain the contribution of deaths at different age groups and from different causes to changes in life expectancy at birth in Czechoslovakia, Hungary and Poland between 1979 and 1990. RESULTS: Improvements in infant mortality have been counteracted by deteriorating death rates among young and middle-aged people, with the deterioration commencing as young as late childhood in Hungary but in the thirties or forties in Czechoslovakia and Poland. The leading contributors to this deterioration are cancer and circulatory disease but, in Hungary, cirrhosis and accidents have also been of great importance. CONCLUSIONS: The patterns observed in each country differ in the age groups affected and the causes of death. Further work is required to explain these differences.

Development of public health training in Hungary--an exercise in international co-operation.

Following the fall of Communism, the countries of Central and Eastern Europe have been restructuring their health systems. Restructured medical education is vital to, and has been supported by, the European Union TEMPUS (Trans-European Mobility Scheme for University Studies) scheme. An example is a three-year project in Hungary to develop undergraduate and postgraduate medical education in public health. The project team (CNPHH) included representatives from all Hungarian medical schools and universities in Western Europe and Canada. The project involved assessing the needs of the Hungarian departments and developing a strategy involving transfer of skills in modern public health and educational methods, study visits and courses in Hungary and at Western universities, and capital funding. The project provides a model for others wishing to develop or restructure their education at national or local levels. This paper describes the methods used, outcomes achieved, and lessons learned.

The electronic medical-student exchange: a low-cost alternative to overseas electives.

The authors report on an international collaboration that uses the Internet for medical education. In addition to introducing the students to electronic communication, the project aims to further international collaboration and understanding and to emphasize the importance of a population perspective for health and disease. Students and professors in Canada, England and Hungary participated.

Teaching public health: an innovative method using computer-based project work.

Restructuring of training in public health in the Hungarian medical schools is being undertaken in the context of a major European Union TEMPUS Joint European Project. Under the aegis of this project a common core curriculum of public health has been developed. As part of the implementation of the curriculum, new approaches to learning are being explored that should enable students to appreciate the nature and magnitude of the major challenges to public health in Hungary and promote the development of their analytic, interpretative and presentational skills. One of the approaches is based on the individual preparation of reports on important public health issues, making use of secondary data from electronic databases (WHO HFA/PC and OECD Health Data) and traditional printed sources (annuals). This method called 'computer-based project work' was introduced in Debrecen in 1992-1993 with a secondary objective to develop basic computing skills. The initial experiences of introducing computer-based project work to the curriculum have been positive. This paper describes a practical example of the implementation of innovative approaches to teaching in a highly traditional setting in Central Europe, and one that provides ideas and encouragement to those facing similar problems in the countries of Central and Eastern Europe and the former Soviet Union.

Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line.

Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.

Establishing an international computer network for research and teaching in public health and epidemiology.

Most universities and major research institutions in North America, Western Europe and around the Pacific are connected via computer communication networks. The authors have used these networks' accessible, low cost, electronic mail system to develop a network of public health researchers and teachers. Current and potential uses of this network are discussed. These networks can not only facilitate international cooperation within public health; they also make it possible to conduct international collaborative research projects that would be too cumbersome and time consuming to initialize and conduct without this communication facility. One participant from Hungary has been able to participate in the network by using telefax. This has some drawbacks compared to electronic mail. In this era of rapid change in Eastern Europe, we urge that electronic communication be made freely available to colleagues in Eastern Europe.

Avoidable mortality. Is it an indicator of quality of medical care in eastern European countries?

Age-standardized time trends (1979-1988) for avoidable mortality in two Eastern European countries (Hungary and Czechoslovakia) and selected developed countries (England and Wales, France, Italy, Japan, Portugal and USA) have been analysed. Mortality from both all avoidable causes and all other causes declined in the selected developed countries during the period of observation, the decline in rates for avoidable causes was faster than that for all other causes. In Hungary and Czechoslovakia the death rates from both groups of causes increased in the first part of the period studied and a decline in mortality from both types of causes could be observed from 1985. As a consequence, the difference in avoidable mortality between the Eastern European countries and the developed countries increased by the end of the observation. Studies on mortality from individual amenable causes showed that the death rates are usually much higher in Hungary and Czechoslovakia than in the developed countries and the differences did not diminish during the period of study. In Hungary and Czechoslovakia the bad pattern of mortality from conditions amenable to medical interventions is believed to reflect, at least in part, the crisis in the health services which these countries have experienced for the past decades.

Cytogenetic analyses of three papillary carcinomas and a follicular adenoma of the thyroid.

Cytogenetic data of three papillary carcinomas and a follicular adenoma using direct preparations or cell cultures or both after 7 to 60 days in vitro are presented. Although karyotype of the follicular adenoma proved completely normal, in each of the three papillary carcinomas a modal chromosome number in the diploid range and a deleted 11q were observed. In case 1 the del(11)(q23) was associated with rearrangement of chromosome 1 and other marker chromosomes. Our results suggest that 11q deletion may be specific for papillary carcinoma of the thyroid.

Cytotoxic endothelial cell antibodies in mixed connective tissue disease.

We examined the presence of anti-endothelial cell antibodies in the sera of 44 patients with mixed connective tissue disease (MCTD). Warm antibodies against endothelial cells were found in 45.4% of patients; the presence of these antibodies was positively correlated with anti-monocyte antibodies (P less than 0.01) but not with anti-lymphocytic antibodies. Strong correlations were found between the presence of these antibodies and abnormalities of pulmonary ventilatory capacity, neurophysiologic and myocardial function. Anti-endothelial antibodies were related to the high rate of spontaneous abortion noted in female MCTD patients. The identification of the antigen identified by MCTD sera in endothelial cells would help in understanding disease manifestations.

Effect of enzymic (collagenase) harvesting on the intracellular Na+/K+ ratio of Swiss/3T3 cells as revealed by X-ray microanalysis.

Swiss/3T3 cell cultures were harvested with 0.05% collagenase and after centrifugation the pellet was prepared by the freeze-fracture/freeze-drying (FFFD) method for bulk-specimen X-ray microanalysis. Time-dependent variations in the intracellular monovalent elemental concentrations (Na+, K+ and Cl-) as well as of the Na+/K+ ratio were followed for 120 min subsequent to harvesting. The quantitative measurements revealed a very considerable increase in the intracellular Na+ and Cl- accompanied by a decrease in the K+ concentration as soon as 5 min after harvesting. The Na+/K+ ratio had increased by this time to about 1.5 on average. These changes indicate a sustained depolarization of the cell membrane. During the first 60 min this depolarization tended to normalize as demonstrated by an exponential decrease in the intracellular Na+ and Cl- and an increase in the K+ content involving a decrease in the Na+/K+ ratio. The total intracellular monovalent ion concentration remained almost constant during this post-harvesting period. These results suggest that harvesting represents a serious depolarizing stimulus to the cells, the consequences of which are restored only after 1-2h. These alterations should be taken into consideration during various experimental designs when using anchorage-dependent cell cultures.

In vivo formation of a carcinogenic substance from diethyl pyrocarbonate in the presence of ammonia.

Mice of different strains have been treated repeatedly with diethyl pyrocarbonate (DEPC) and/or ammonia by gastric tube. In adult mice treated with DEPC plus ammonia, pulmonary tumours developed. However, DEPC or ammonia alone proved not to have any carcinogenic effect. When DEPC administration was followed by ammonia treatment more pulmonary tumours developed than in the case of ammonia-DEPC sequence. The shorter the time interval between DEPC and ammonia administrations the higher the number of lung tumours observed. Pulmonary tumours could not be observed in the offspring of pregnant mice treated with DEPC and ammonia or in suckling mice whose mother was treated with DEPC and ammonia. In gastric juice a new substance is formed from DEPC in the presence of ammonia. This new substance was very similar or identical to urethane according to the thin layer chromatographic investigation.

Effect of 12-O-tetradecanoylphorbol-13-acetate on selection of drug-resistant phenotypes in rodent cells.

The effect of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the selection of drug-resistant phenotypes in hamster and mouse cells has been studied. TPA increased considerably the incidence of methotrexate (MTX)-, N-(phosphonacetyl)-L-aspartate- and cadmium-resistant 3T6 and 3T3 mouse cell clones, but it had little effect on V79 and CHO hamster cells. The MTX-resistant V79 hamster cell clones, selected with or without TPA, lost their resistance within 15 cell cycles, however, 50% of the 3T6 mouse cell clones selected and maintained in the presence of TPA preserved their resistance for more than 15 cell cycles. A number of the MTX-resistant 3T6 clones overproduced dihydrofolate reductase enzyme; however, their frequency was lower among the clones selected in the presence of TPA.

Effect of tumor promoter 12-O-tetradecanoylphorbol-13-acetate on recovery of methotrexate-, N-(phosphonacetyl)-L-aspartate-, and cadmium-resistant colony-forming mouse and hamster cells.

The effect of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and its nontumor promoting derivative 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate on the frequency of mouse and hamster cells resistant to methotrexate (MTX), N-(phosphonacetyl)-L-aspartate, and cadmium has been examined. TPA alone at concentrations up to 1.0 microgram/ml had no significant effect on the plating efficiency of either mouse or hamster cells. Exposure of 3T3 and 3T6 mouse and V79 and Chinese hamster ovary cells at low density to the 3 compounds in the presence of TPA (0.1 microgram/ml) did not result in any increase in the recovery of resistant colonies. When plated at high density, exposure to drug selection in the presence of TPA resulted in a 3- to 10-fold increase overall in the incidence of MTX-, N-(phosphonacetyl)-L-aspartate-, and cadmium-resistant mouse cells. However, an increase greater than 3-fold was not observed in hamster cells exposed to drug plus TPA under the same conditions. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate had no significant effect on the frequency of MTX-resistant cells. Seventy V79 cell clones surviving MTX (200 to 400 nM) alone and 79 surviving MTX plus TPA were isolated and retested for resistance to MTX. None were stable. In contrast, 6 out of 42 mouse colonies isolated from MTX alone and 55 out of 99 isolated from MTX plus TPA showed stable resistance on retesting in MTX. The implications of these results in relation to possible mechanisms of tumor promotion are discussed.

Effects of urethan on lymphokine-producing activity of lymphocytes and on some functions of peritoneal macrophages in rats.

Effects of urethan on some functions of blood lymphocytes and peritoneal macrophages (PMs) of rats were studied in in vivo and in vitro experiments. The in vitro lymphokine (LK) producing activity of lymphocytes in the presence of specific antigen was depressed by urethan administered 1-5 days before the BCG sensitization. However, the drug injected after the BCG sensitization was not effective on the LK production. Urethan added to the cultures of previously BCG-primed lymphocytes did not influence the LK production. The sensitivity of glycogen-provoked PMs (pPM) to LK-induced activation and, at the same time, the 125I-IgG2a binding capacity as well as the EA rosette formation of the provoked PMs were depressed by urethan administered 1-5 days before the lavage of PMs. These functions of resident PMs (rPM) were not altered by the drug treatment. Urethan added to the cultures of resident or provoked PMs proved to be ineffective. These results led to the conclusions that urethan, after its in vivo metabolic conversion, causes an impairment of the macrophage functions in the inductive phase of the immune response and this event may be the crucial point in the immunosuppressive effect of urethan.