Transmembrane chloride flux is required for target cell lysis but not for Golgi reorientation in cloned cytolytic effector cells. Golgi reorientation, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase release, and delivery of the lethal hit are separable events in target cell lysis.

Cell-mediated cytotoxicity can be inhibited by the replacement of chloride with ions that are incapable of passing through chloride channels or by the presence of stilbene disulfonate derivatives known to interfere with chloride flux. We show that the stilbene disulfonate (4,4-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibits lysis of YAC-1 targets by the cloned cell line NKB61A2. Inhibition of lysis occurs on the level of the effector cell inasmuch as preincubation of effectors but not of targets interferes with subsequent lysis. Moreover, inhibition of chloride flux in the target does not interfere with target cell lysis by cytotoxic granules isolated from killer cells. Target cell binding takes place in the presence of DIDS or absence of external chloride, suggesting that events that follow target cell binding require chloride flux. We show that reorientation of the Golgi apparatus, which occurs subsequent to target cell binding in the effector cell, occurs under conditions that interfere with chloride flux. It is therefore suggested that events in the effector cell taking place subsequent to the Golgi apparatus reorientation reaction are inhibited and that delivery of the lethal hit is a stimulus-induced secretory event that requires transmembrane chloride flux. Delivery of the lethal hit is shown to be independent of the release of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase, suggesting that cytolytic components and BLT serine esterase are likely packaged in different vesicles.

High activity of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and cytolytic perforin in cloned cell lines is not demonstrable in in-vivo-induced cytotoxic effector cells.

Recent observations have suggested striking similarities between complement-mediated and cell-mediated lysis. Both pathways share the terminal insertion of channels into target membranes, and unique esterases have been postulated to participate in the activation of cytolytic effector molecules. Since killer-specific esterases and channel-forming proteins can be demonstrated in in vitro cell lines, it is important to ascertain that the described esterase and channel-forming proteins are also present in killer cells from in vivo sources. Results presented here show that killer-cell-specific N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase is induced in vitro concomitant with the sensitization of cytotoxic effector cells. In contrast, in-vivo-primed cytotoxic T cells or natural killer (NK) cells fail to express high levels of this enzyme. Assay of different cytotoxic effector cells reveals the presence of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase in clones with T killer and NK activity, but enzyme levels do not correlate with cytolytic activity nor does inhibition of esterase activity interfere with granule-mediated cell lysis. A similar result is seen with granule-mediated cytolytic activity. Cloned NK and T killer cell lines possess granules that are able to lyse erythrocyte targets. However, T killer cells sensitized in mixed lymphocyte culture or in vivo have no detectable cytotoxic granules. Cytotoxic granules are also not detected in NK cells isolated from animals.

Histocompatibility antigens in leprosy.

Results of HL-A typing are presented in 82 patients with leprosy and 50 normal Filipinos from Cebu, and 144 normal Filipino immigrants from the Luzon area. Comparisons of HL-A antigen frequencies among the total patients and normals of Cebu showed no statistically significant differences; however, HL-A10 was increased in frequency among the patients with lepromatous disease compared to the normals, and HL-A5 was increased among the tuberculoid patients compared to the lepromatous patients. None of these comparisons was statistically significant when corrected for the number of antigens tested. Comparisons of HL-A antigen frequencies between normal Filipinos of the Cebu and Luzon regions showed increased W-5 in the Luzon population (corrected p smaller than 0.025).

Calorific content of certain bacteria and fungi.

Calorific contents of dried cells of several representative species of bacteria (gram-negative rods and gram-positive rods and cocci), two species of yeasts, and a filamentous fungus were determined by bomb calorimetry. The grand mean was 5,383 cal per g of ash-free dry weight. This value was then used to determine quantity of energy assimilated (E(a)) during growth. Subsequently, E(a) was employed in the equation: Y(kcal) = Y/(E(a) + E(d)), where Y(kcal) is the yield of cells per kilocalorie of energy taken from a culture medium, Y is the yield per mole of substrate utilized, E(a) is Y times caloric content of the cells, and E(d) is the energy expended by oxidative dissimilation. An estimate of E(d) was obtained for a number of experiments by multiplying the moles of oxygen consumed per mole of substrate utilized during growth by the average quantity of energy utilized to reduce a mole of oxygen with electrons from organic compounds (106 kcal). From previous studies in our laboratories, a value for Y(kcal) of 0.118 g/kcal was predicted. The mean value for data from five studies of aerobic growth of prototrophic heterotrophs was found to be 0.111.

Growth yields of bacteria on selected organic compounds.

Cell yields were determined for two bacterial soil isolants grown aerobically in minimal media on a variety of synthetic organic compounds. 1-Dodecanol, benzoic acid, phenylacetic acid, phenylglyoxylic acid, and diethylene, triethylene, and tetraethylene glycols were tested. Two "biochemicals," succinate and acetate, were also tested for comparison. Yields were calculated on the basis of grams of cells obtained per mole of substrate utilized, gram atom of carbon utilized, mole of oxygen consumed, and equivalent of "available electrons" in the substrates. This latter value appears to be nearly constant at 3 g of cells per equivalent of "available electrons." Yields predicted on this basis for other bacteria and for yeasts on other substrates are in fair agreement with reported values.