Kenneth R. Rozee

Production of Interferon by Peripheral Blood Mononuclear Cells from Normal Individuals and Patients with Chronic Lymphocytic Leukemia

Peripheral blood mononuclear cells (PBMC) from normal individuals were studied to identify which cells produce alpha-interferon (IFN-alpha) in response to a virus stimulus. It was found that cells both adherent and nonadherent to plastic formed IFN-alpha after induction by any one of several viruses studied. When nonadherent cells were separated on discontinuous Percoll gradients, only the cells in the less dense Percoll fractions produced IFN, whatever the virus used. By indirect immunofluorescence with monoclonal antibodies to HLA-DR and to Leu 11b, the distribution of the HLA-DR+ cells was shown to resemble most closely that of the IFN-producing population. Elimination of these cells (by complement-mediated lysis with the same antibodies) abrogated the IFN response, but NK cells remained and thus do not produce IFN-alpha. In confirmation, elimination of the Leu 11b+ cells had no effect on the amount of IFN produced. PBMC preparations from patients with chronic lymphocytic leukemia (CLL) appeared incapable of producing IFN-alpha but were shown to contain identifiable IFN-producing cells. The low or absent IFN levels in CLL are probably due to the relative scarcity of IFN-producing cells in their PBMC.

INSECTICIDE AND VIRAL INTERACTION AS A CAUSE OF FATTY VISCERAL CHANGES AND ENCEPHALOPATHY

The possibility of an ecological interaction by chemical “priming” of young animals for viral infectivity stimulated this investigation.1650 one day old mice were given topically applied chemical dissolved in corn oil for eleven days: (a) DDT, (b) organophosphate (commercial Fenitrothion), (c) organophosphate plus DDT, (d) corn oil alone. Twenty-four hours after discontinuance of all chemicals, 0.05 ml. (sublethal dose) of E.M.C. virus was injected subcutaneously in known titers.Mortality rates in a 12 day period following injection were (a) 0-10% in DDT groups, (b) 3-8% in organophosphate groups, (c) 72-100% in organophosphate plus DDT groups, (d) 0-5% in corn oil groups.Pathologically, fatty degeneration was noted in the insecticide/viral groups. The encephalopathy showed no specific CNS pathology but death followed a sequence of paralysis and convulsions. Similar susceptibility with PCBs plus virus was reported in young ducks by Friend and Trainer (Science, 170:1314, 1970).The effect of currently used combinations of insecticides on human viral susceptibility requires further attention.

The properties of emulsifiers that enhance the replication of viruses in cell cultures

Abstract Some of a variety of commercial emulsifiers were capable of enhancing the sensitivity of mammalian cells in culture to infection with several viruses. Those viruses which were enhanced in their replication were single-stranded RNA viruses whereas the double-stranded viruses tested were non-responders. In addition, some cells lines, as compared to others, responded in a more sensitive manner to the active emulsifiers.

BIOLOGICAL CHARACTERIZATION OF TUBERCULIN‐INDUCED HUMAN LYMPHOCYTE INTERFERON‐LIKE SUBSTANCE (PPD‐IF)

We described (1) an in vitro PPD-induced interferon (IF) system using human peripheral lymphocytes producing acid-labile (type 11) human IF; (2) the immunocyte origin of this IF and (3) the antiviral and the cell-multiplication-inhibitory activities of type I1 IF compared to classic acid-stable (type I) human leukocyte IF (Leu-IF) in various cell cultures. Leukocytic cell suspensions were prepared from peripheral blood obtained from groups of healthy Mantoux positive (PPD-POS) and negative (PPD-NEG) adults. PPD sensitivity, assessed by skin test reactivity, was correlated with the donors’ lymphocyte antigenic response to PPD (2-25 pg/ml), using uptake of H3-TdR into DNA’ on day 6 of culture. Peripheral blood mononuclear cells (PBMC) were isolated from 50-100 ml of heparinized venous blood by Ficoll-Hypaque (F/H) gradient centrifugation.* PBMC were further separated by repeated rosetting with neuraminidase-treated sheep erythrocyte (El followed by F&H centrifugation into E-rosette-forming (E-RF) (T-enriched) and non-E-RF (B-enriched + monocyte) cell populations? Monocytes were separated from the B-enriched cell fraction by adherence to plastic surface (Falcon dish) and by subsequent recovery with a rubber policeman. The purity of T-enriched, B-enriched and monocyte populations was assessed by E-RF, representing T cells; by immunofluorescence staining of surface immunoglobulin and by EAC rosette formation,’ a characteristic of B cells: and by peroxidase staining reactivity, a property of mon~cytes.~ Cell viability was assayed by trypan blue exclusion and was greater than 98%. The functional capacity of T cells was monitored by their 3-day mitogenic response to PHA, and of B cells by their %day mitogenic response to PWM when cultured together with puromycin-treated T cells.3sE IF production experiments were done in 1 ml volume of McCoy’s 5a medium plus 15% heat-inactivated fetal calf serum and antibiotics in 16 x 150 mm plastic tubes (Falcon 2045) containing 2 x 10’ lymphocytes; 0.1 ml amounts of PPD (Central Veterinary Laboratory, Weybridge, Surrey, England) were added to each culture at the desired concentration. IF assay was performed on B-SC-1 cells grown in 24-well Cluster plates (Costar). The IF assay was performed on B-SC-1 cells grown in 24-well Cluster plates (Costar). The IF titer was expressed as the reciprocal of that dilution of sample, which, in 0.5 ml volume, resulted in 50% reduction of plaques produced by bovine vesicular stomatitis virus (PRDSO unit) (U). The results of experiments using PBMC from two PPD-POS donors with PPD at different concentration revealed a concentration-dependent IF response. Cultures incubated with PPD at 50, 100, 250, 500 and 1000 pg/ml, produced a significant IF response (12-270 U) throughout a 6-day period. Peak IF response (270 U) on day 3 occurred in cultures exposed to 250 pg of PPD without an

636 ACETYLSALICYLIC ACID (ASA) AND ACETAMINOPHEN (AAP) AS POSSIBLE CONTRIBUTORY FACTORS IN THE ETIOLOGY OF REYE'S SYNDROME

A mouse model of Reyes syndrome (RS), in which exposure to an emulsifier prior to Influenza B virus infection induced most of the biochemical and histologic features of human RS, was used to investigate ASA and AAP as initiators and enhancers of this syndrome. Mortalities of mice increased when ASA and AAP was given to 2 week old mice previously exposed to emulsifier and Influenza B (p=0.02 and p=0.001 respectively). In 5 week old mice, mortalities increased in mice infected with ASA coincident with administration of the drug but independent of the emulsifiers (p=0.01). In vitro work with MDCR (dog kidney) cell cultures were used to study the cytotoxic effect of ASA and AAP on Influenza B infection. Neither ASA or AAP, up to cytotoxic concentration, had any effect on the sensitivity of MDCR cells to Influenza B infection. Both drugs, however, substantially reduced the sensitivity of these cells to exogenously provided interferon. As both drugs are mitochondrial toxins and displayed similar interactions with the virus, these studies question the substitution for ASA in young patients with influenza infection.

997A A COMPROMISED INTERFERON RESPONSE: AN ETIOLOGIC FACTOR IN REYE'S SYNDROME

Young mice treated with Toximul MP8, a polyoxyethylene ether based emulsifier, show an increased mortality when infected with encephalomyocarditis virus (EMC) than do control mice. Lymphocytes taken from these emulsifier treated mice respond poorly to interferon induction, as compared to controls. Interferon also protects control mice against EMC infection but such protection is reduced in emulsifier-treated mice. This enhanced lethality to EMC in emulsifier-treated mice may be due to compromised interferon response in these animals.Blood samples were obtained from a group of Reyes patients in the acute and convalescent phase of their disease. Lymphocytes were induced to synthesize interferon by Newcastle Disease Virus. Peripheral blood lymphocytes from convalescent patients and controls responded well to interferon induction. Lymphocytes taken in the acute phase of children with Reyes syndrome produced significantly less interferon than those from recovering patients or controls. Since Reyes syndrome is an unusual response on the part of the host following a virus infection our data would appear to implicate interferon as part of this altered response.

536A AN EXPERIMENTAL MODEL OF REYE'S SYNDROME: CHEMICAL AND INFLUENZA B VIRUS INTERACTION

In this study, we describe an experimental model using a mouse-adapted strain of Influenza B (Inf. B) and a surfactant (Toximul MP8) known to enhance infection with encephalomyo-carditis virus in mice. One of the theories of etiology of Reyes syndrome (RS) is that an environmental toxin predisposes the young child to react abnormally to a virus infection. Inf. B is the most common virus associated with RS and its substitution into this model would have more relevance. Suckling mice were injected i.p. with a non-lethal dose of Toximul MP8. At various time intervals after chemical exposure the mice were infected intranasally with sub-lethal doses (less than 1 LD50) of Inf. B. Mice exposed to a combination of chemical and virus showed a much higher mortality as opposed to control groups. This enhancing effect of Toximul MP8 was found to be time dependent. The brains and livers of animals show no cellular infiltrate and the livers showed various degrees of fine fatty changes. Urea cycle enzymes (ornithine transcarbamylase, carbamyl phosphate synthetase) were reduced. The cytoplasmic enzymes of the urea cycle were unchanged. This RS experimental model will allow the study of therapeutic intervention and the pathophysiological events leading to this fascinating syndrome.

LETHAL VIRUS-EMULSIFIER|[sol]|SOLVENT INTERACTION: IN VIVO AND IN VITRO STUDIES

Chemical emulsifiers and solvents are widely used in industrial compounds as dispersal or wetting agents. These compounds, which include insecticide spray formulations, are diverse alkylated aromatic molecules largely derived from petroleum oil byproducts. Previous experiments (Science, 192, pp. 1351-1353, 1976) have indicated increased lethality of EMC virus-infected young mice pre-exposed to several of these compounds. Further experiments with young mice using the emulsifiers Toximul, and Atlox, and the solvent Aerotex, have shown each enhances, to varying degrees, the lethality of EMC in suckling mice.Similarly various tissue culture systems (Hela, L-929, VERO, and secondary human kidney cells) were pretreated with subtoxic concentrations of the chemicals (0.01-10 ppm) and subsequently infected with appropriate viruses (VSV, EMC, Polio Type I). The results indicate that these chemicals significantly potentiate the plaquing efficiency of the culture systems.The potentiating effect of these chemicals on certain viral infections has thus been demonstrated both in vivo and vitro.The manner in which these complex chemicals alter a hosts reaction to a virus may be important in the pathogenesis of such conditions as Reyes syndrome.