Effect of cortisol on the growth of Chlamydia trachomatis in McCoy cells.

The number of intracytoplasmic inclusions of Chlamydia trachomatis produced in McCoy cell monolayer cultures infected with a constant inoculum of a recently isolated genital strain was compared in cultures of untreated replicating cells and in monolayers which had been incubated in the presence of cortisol at initial extracellular concentrations between 0.0001 and 100 microgram/ml. The effect of adding cortisol was dependent on its concentration, on the time of addition to the tissue culture medium, and on the initial number of McCoy cells seeded to form the monolayer. When a concentration of 1.0 microgram/ml was added at the time of infection with C. trachomatis, the number of inclusions detectable after a further 48 h of incubation was increased by 1.84-fold over those detected in untreated cells. The mean size of inclusions and the ease of their recognition in McCoy cell cultures was also increased by this procedure.     

A selection system specific for the thy mutator phenotype

Thy^– mutants, in addition to being resistant to arabinosyl cytosine (arcC), show cross-resistance to 5-fluorouracil (5FU). When Chinese hamster ovary (CHO) cells were exposed to a selection system using both araC and 5FU, the resistant clones isolated were identical to thy^– mutants by the following criteria: (1) all were auxotrophic for thymidine with a high reversion frequency to thymidine prototrophy; (2) those tested had a high level of dCTP relative to wild-type cells, while dTTP and dATP levels were unaffected; and (3) all tested had a 7- to 50-fold higher rate of spontaneous mutation than the wild-type strain for at least one independent genetic marker. Although spontaneous thy^– mutants were rare, the frequencies of thy^– mutants in untreated and mutagenized cultures are consistent with the conclusion that the thy^– phenotype is the consequence of a single mutation in CHO cells.     

Growth of thePhlebovirus Toscana in a mosquito(Aedes pseudoscutellaris) cell line (AP-61): Establishment of a persistent infection

Summary Toscana Virus, a sandfly-associatedPhlebovirus, was adapted to grow in culturedAedes pseudoscutellaris (AP-61) mosquito cell line. No evidence of virus growth was seen after primary infection of cell monolayers under maintenance conditions. On the contrary, persistent infections were established by subculturing infected cultures. Cytopathic effect was never observed. Significant titres of virus (10³–10⁵ PFU/ml), as assayed in Vero cells at 37° C, were released from persistent infected cells after several subcultures at 29° C over a period of six months. The percentage of virusproducing cells in the persistently infected cultures varied from 0.003 to to 0.017 per cent, whereas from 10 to 50 per cent of cells were shown to retain viral antigens by immunofluorescence. The virus released from persistently infected cultures did not show changes in both plaque size and temperature sensitivity from the parental virus. The virus released from persistently infected cultures multiplied in AP-61 cell monolayers reaching relatively high titres (10⁴ PFU/ml).With 5 Figures     

DNA labeled during phosphonoacetate inhibition and following its reversal in herpesvirus infected cells

Summary Human embryonic lung cells were pre-equilibrated with phosphonoacetate and³²P orthophosphate label, then infected with phosphonoacetate-sensitive herpes simplex virus (HSV) type 1. Analyses of viral DNA produced in these cells showed the following. i) Viral DNA was synthesized in infected cells exposed to 100 µg of the drug per ml of medium but not in cells exposed to four-fold higher concentrations of the drug. ii) At 300 µg/ml a region of the DNA between 0.58 and 0.69 map units became transiently labeled, but the restriction endonuclease fragment containing these sequences migrated more slowly than the corresponding fragment from virion DNA. iii) Viral DNA extracted from infected cells 1.5 hours post drug withdrawal (300 µg/ml) was preferentially labeled in 2 regions of the genome mapping between 0.17 and 0.23 and 0.58–0.69 map units. This finding is in agreement with a report ofFriedman et al. (8) suggesting that HSV DNA contains two different sites of initiation. In addition a 4.8×10⁶ molecular weight fragment was also preferentially labeled. This fragment could represent a smaller, aberrantly migrating fragment from the 0.17–0.27 map unit region of the DNA. (iv) Viral DNA extracted from infected cells at longer intervals after drug withdrawal showed an increasing gradient of radioactivity progressively labeling the genome. These results are consistent with the hypothesis that viral DNA has at least two sites of initiation of DNA synthesis and that both sites are within the L component of the DNA. Alternatively, the results could be interpreted as two sites of localized synthesis (repair) that are detected at high concentrations of phosphonoacetate and immediately following reversal of inhibition of DNA synthesis. The results do not exclude the possibility that secondary sites in both L and S are utilized late in infection or in untreated cells.With 7 Figures     

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2× L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.     

Die Isolierung von Poliovirus in verschiedenen Gewebekulturen

Summary 100 stool extracts from patients with poliomyelitis or from individuals after oral poliomyelitis vaccination were inoculated simultaneously into HeLa, monkey kidney, and human thyroid cultures. In most cases (88 times) a cytopathic effect was seen in all three cultures. Failures were most frequently encountered in thyroid, rarely in monkey kidney cells. They were due either to thyroid culture lots with reduced viral susceptibility or to small amounts of virus in the stool specimens. No qualitative difference was found in the host range of individual virus strains. With attenuated viruses the cytopathic effect in HeLa cells as a rule was delayed for several days.

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Die Untersuchungen wurden mit Unterstützung der Deutschen Vereinigung zur Bekmpfung der Kinderlähmung e.V. durchgeführt.

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Wir danken Herrn Privatdoz. Dr. F.Müller, Düsseldorf, für die freundliche überlassung einiger virushaltiger Stuhlproben. Herr Dr. A. R.Ababio, Homburg, war uns bei der Beschaffung von Stuhlproben nach oraler Poliomyelitisimpfung behilflich. Wir danken schließlich Fräulein E.Baschleben, Frau H.Gelderblom, Frau H.Kaiser und Fräulein I.Lamy für ihre Hilfe bei den Untersuchungen.     

Sugar transport in isolated rat kidney papillary collecting duct cells

d-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry ofd-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate ofd-glucose uptake was measured indirectly by monitoring thed-glucose-dependent O₂ uptake in the presence of the uncoupler CCCP.d-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10^–3 M. Uptake was, however, completely inhibited by 10^–5 M cytochalasin B and 10^–4 M phloretin. The apparentK _i for cytochalasin B was 1.5×10^–6 M and for phloretin 2.0×10^–5 M. Studies on the substrate specificity revealed that at 1 mMd-mannose is taken up and metabolized to the same extent asd-glucose. A 50-fold higher concentration of 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose inhibitedd-glucose uptake completely whereas -methyl-d-glucoside,d-allose, andd-galactose were without effect. Under conditions whered-glucose utilization was maximally stimulated an apparentK _m of 1.2 mM and aV _max of 1 mmold-glucose/g protein hour was found ford-glucose uptake.These results indicate that thed-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of pelarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.Supported by grant DFG Gr 916/1-1     

Increased resistance of SV40 transformed human amnion cells to poliovirus infection

Summary The increased resistance to poliovirus of SV₄₀ transformed human amnion cells compared with primary cells has been shown to involve reduced cytopathic effect (CPE), delayed and reduced production of virus, inefficiency of infectious center formation, and the failure of transformed cell monolayers to support poliovirus plaque formation. At multiplicities of exposure (plaque-forming units/ cell) less than 10, CPE was negligible in the poliovirus infected transformed cell cultures which could be maintained as living cultures yielding poliovirus up to three months. Infectious center formation at different multiplicities showed that both cell types could be infected with single infectious units, but in cultures of transformed cells, the probability of initiating infection was reduced about 25-fold. Resistance was not observed in primary amnion cultures infected with SV₄₀ in the absence of transformation. However, cellular resistance developed as morphologic transformed cells appeared in SV₄₀ infected cultures. Transformed cells maintained resistance when SV₄₀ multiplication in transformed cultures was suppressed with metabolic inhibitors or eliminated by isolating virus-free clones of transformed cells in the presence of SV₄₀ antiserum.The mechanism of the increased resistance of SV₄₀ transformed human amnion cells to poliovirus infection involves an inefficiency in the initiation of infection. When compared to the highly susceptible untransformed primary cells, no significant differences in the adsorption, penetration or eclipse of poliovirus were demonstrated. There was no detectable interferon production in the transformed cultures either before or after poliovirus infection. Since infection with infectious poliovirus RNA bypassed resistance, it is concluded that the resistance of the SV₄₀ transformed cells results from an inefficiency in the uncoating of poliovirus to infectious RNA.This investigation was supported in part by NCl Research Grant CA-08748. Part of thesis submitted byEdwin Hahn to Cornell Graduate School of Medical Sciences, Cornell University, New York, New York, for the degree of Doctor of Philosophy.     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

μ and δ opioid receptor activation inhibits ω-conotoxin-sensitive calcium channels in a voltage- and time-dependent mode in the human neuroblastoma cell line SH-SY5Y

 Ca²⁺ channel modulation by the μ opioid agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAGO) and the δ opiate agonists [d-Pen², d-Pen⁵]-enkephalin (DPDPE) and [d-Ala², d-Leu⁵]-enkephalin (DADLE) in cultured human neuroblastoma SH-SY5Y cells was investigated using the whole-cell variant of the patch-clamp technique. In SH-SY5Y cells, differentiated in vitro with retinoic acid, all agonists reversibly decreased high-voltage-activated, ω-conotoxin-sensitive Ba²⁺ currents in a concentration-dependent way. Inhibition was maximal with a 1 μM concentration of opiate agonists (76% with DAGO and 63% with δ agonists, when measured at 0 mV) and was characterized by a clear slow down of Ba²⁺ current activation at low test potentials. Both inhibition and slow down of activation were attenuated at more positive potentials, and could be partially relieved by strong conditioning depolarizations. Current suppression operated by both μ and δ agonists was prevented by pre-treatment of the cells with pertussis toxin. No sign of additivity was observed when δ agonists were applied to cells that were maximally activated by DAGO, suggesting that a common mechanism, involving the same type of modulating molecule, is responsible for Ca²⁺ channel inhibition promoted by activation of μ and δ opioid receptors in SH-SY5Y cells. Received: 10 October 1996 / Received after revision and accepted: 18 November 1996     

Über die Virusempfindlichkeit von Schilddrüsen-Zellkulturen

Summary Infected tissue culture fluid of prototype strains of various viruses was passed five times in human thyroid tissue cultures. Comparative titrations were carried out with both original culture fluid and virus from the fifth cell passage in thyroid cells as well as in HeLa or monkey kidney cells respectively. By this means thyroid cells were found to exhibit an equal (or in some instances higher) susceptibility as the culture of origin to vaccinia virus, herpes simplex virus, parainfluenza viruses types 1, 2 and 3, adenoviruses types 3, 6, 9 and 12, two prototype strains of Coxsackie B3 virus, Coxsackie A9 virus, ECHO 9 virus [prototype Hill and an epidemic strain (Russ.)], an attenuated and a virulent strain of each of the three poliovirus types. The virus yield in thyroid and the original cell system was found to be in the same range. ECHO 16 virus, two prototype strains of Coxsackie B4 virus and B.V.A. 96 strain of Coxsackie B2 virus produced no cytopathic effect in thyroid cell cultures and no inapparent multiplication was demonstrable. The Ohio 1 strain of Coxsackie B2 virus had a somewhat lower infectivity for thyroid than for monkey kidney cells. Reovirus type 1 showed minimal cytopathic change and only a slight multiplication in thyroid cells. From the ECHO virus group, the prototype strains of the types 1, 2, 3, 5, 6, 7, 8, 11, 12, 13, 19, 20, 22 and 24 could be passed readily with CPE in thyroid cells, whereas ECHO types 18 and 23 were erratic and type 17 produced no CPE. The prototype strains of adenovirus types 1 to 28 as well as 24 other strains of various serotypes were cytopathic for thyroid cells without exception. Our experience with the culture and maintenance of human thyroid cells as obtained in the course of this study has been reported.

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Die vorliegenden Untersuchungen wurden mit Unterstützung der Deutschen Vereinigung zur Bekämpfung der Kinderlähmung e. V. und der Deutschen Forschungsgemeinschaft durchgeführt. Einer von uns (A. R. A.) verdankt dem Ghana Cocoa Marketing Board ein Stipendium. Die Schilddrüsen erhielten wir durch die Freundlichkeit von Herrn Prof. Dr.Lüdeke, Chirurgische Universitätsklinik, Homburg, von Herrn Chefarzt Dr.Schäfer, Kreiskrankenhaus St. Ingbert, und von Herrn Oberarzt Dr.Schrauf, Bürgerhospital Saarbrücken. Ganz besonders danken wir Fräulein E.Baschleben, Fräulein I.Lamy und Frau H.Huwer für ihre technische Hilfe bei den Untersuchungen.     

Growth and cytopathogenicity of Trichomonas vaginalis in tissue cultures.

The primary purpose of this study was to identify the mammalian tissue cultures which were most suitable for investigations of the cytopathogenicity of Trichomonas vaginalis. A recently isolated strain of the organism was inoculated into 15 different tissue cultures which were maintained in an appropriately modified growth medium. Proliferation of the protozoon was accompanied by the progressive disintegration of cell culture monolayers. Initial focal lesions consisting of detached cells and an accumulation of trichomonads gradually enlarged until the entire monolayer was disrupted. When judged by the size of the inoculum required to obtain this effect, differences among the tissue cultures were noted. An inoculum of approximately 10(3) viable trichomonads was sufficient to completely disrupt monolayers of HeLa 229, HeLa, McCoy, HEp-2, and RK-13 cells. To obtain a comparable effect with other cells, 10- to 100-fold higher levels of inoculum were required. Polyethylene glycol concentrates from culture filtrates contained a cell-detaching factor (CDF) which caused detachment and clumping of susceptible cells. Freshly seeded cells in growth medium containing CDF failed to form a monolayer. Aggregates of cells maintained for up to 1 week in the presence of CDF remained viable and formed a monolayer after being washed and suspended in normal growth medium. The activity of the CDF was not lost during 1 week of contact with the cells. The CDF may contribute to the pathogenicity mechanisms of T. vaginalis.     

Graft reaction in tissue culture: II. Quantification of the lytic action on mouse fibroblasts by rat lymphocytes sensitized on mouse embryo monolayers

A method for assaying the lytic action of large pyroninophilic cells (LPC) in cultures of rat lymphocytes on target fibroblasts in mouse embryo monolayers has been described. The basis of the assay was the labelling of fibroblasts with ⁵¹Cr. The rate of ⁵¹Cr release to the medium was found to be proportional to the number of LPC and expressed precisely the actual number of lysed fibroblasts. The lytic activity in LPC suspension was expressed by the lysis index which is the number of LPC per fibroblast lysed at 50 per cent lysis. A suspension of LPC collected 5–6 days after exposure of rat lymphocytes to the mouse monolayers showed a lysis index ranging from 0.7 to 1.3 after 16–48 hours of incubation. The lytic power of a culture was expressed by a value which was obtained by dividing the total number of LPC in a culture by the index. This determined the total number of fibroblasts lysed if the whole culture content were distributed among plates of test monolayer in such a way as to produce 50 per cent lysis in all the plates. Cultures started with 25 × 10⁶ to 30 × 10⁶ lymphocytes produced on the 5th and 6th day a lytic power of as high as 6 × 10⁶ to 12 × 10⁶ fibroblasts. The study has indicated that rats injected with horse serum and boosted 2 days prior to culturing were completely unable to produce graft reaction cultures. Lymphoid cells from non-boosted rats did generate LPC with lytic ability. A quantitative study of the specificity of the lytic reaction has indicated that LPC originated on C57BL/6 monolayers lysed much more strongly monolayers isologous to originator than C3H monolayers, and vice versa.     

Glucose homeostasis across human airway epithelial cell monolayers: role of diffusion, transport and metabolism

Glucose in airway surface liquid (ASL) is maintained at low concentrations compared to blood glucose. Using radiolabelled [³H]-d-glucose and [¹⁴C]-l-glucose, detection of d- and l-glucose by high-performance liquid chromatography and metabolites by nuclear magnetic resonance, we found that glucose applied to the basolateral side of H441 human airway epithelial cell monolayers at a physiological concentration (5 mM) crossed to the apical side by paracellular diffusion. Transepithelial resistance of the monolayer was inversely correlated with paracellular diffusion. Appearance of glucose in the apical compartment was reduced by uptake of glucose into the cell by basolateral and apical phloretin-sensitive GLUT transporters. Glucose taken up into the cell was metabolised to lactate which was then released, at least in part, across the apical membrane. We suggest that glucose transport through GLUT transporters and its subsequent metabolism in lung epithelial cells help to maintain low glucose concentrations in human ASL which is important for protecting the lung against infection.     

Transduction in vivo

Conclusions Transductions and lysogenic conversions were obtained in live chick embryos and in mice. Transductions were successful when lysates were inoculated with or without 30 minutes previous incubation together and also when cultures were inoculated first and lysates 5 hours later.Grateful acknowledgement is made to Professor C. P.Beattie for the help and encouragement given, to Dr.Joan Taylor for confirming the antigenic formulae of some of the organisms, to Dr. A.Bernstein, Dr. F.Kauffmann and Dr. C. C.Spicer for supplying cultures and phage.     

Chemically, mechanically, and hyperosmolarity-induced calcium responses of rat cortical capillary endothelial cells in culture

 The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca²⁺]_i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180–340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, l-glutamate, serotonin, carbachol, noradrenalin, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamin showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca²⁺]_i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca²⁺]_i. Received: 21 September 1998 / Accepted: 8 February 1999     

Evidence for gene silencing by DNA methylation in normal human diploid fibroblasts

Human diploid fibroblasts, strain MRC-5, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) in the S phase of the cell cycle. The frequency of TG^RHPRT^– cells was increased up to 20-fold in comparison to control untreated cultures. Representative TG^R clones were unable to grow in HAT, and these were treated with 5-azacytidine (5-aza-CR). In many cases subsequent growth in HAT medium was observed, but in others it is likely that the cells had run out of growth potential. The results provide the first evidence of the silencing and reactivation of a gene in normal diploid mammalian cells.     

Action of diethylcarbamazine citrate on protective immunity in rats infected withNippostrongylus brasiliensis

Rats made immune toNippostrongylus brasiliensis and treated with diethylcarbamazine citrate (DEC) orally (250 mg/kg×6) exhibited significant suppression of functional immunity. Similarly, administration of compound 48/80 (100 g/rat i.p.) made the immune rats susceptible to challenge infection. Treatment of rats, with 22-day infection with compound 48/80, histamine (20 mg/rat, per os), orl-histidine (20 mg/rat, orally s.c.) did not accelerate worm expulsion. A massive complement-dependent adherence of peritoneal cells (1×10⁸), isolated from immune DEC-treated and untreated rats, to infective larvae (L₃) was observed. Likewise, heavy congregation of normal peritoneal cells to larvae was noticed when the cells were incubated with sera obtained from immune, DEC-treated or untreated rats. The rats receiving mesenteric lymph node cells (125×10⁶ i.v.) or sera (0.5 ml or 1 ml×3 i.p.), obtained from immune DEC-treated rats and challenged with infective larvae developed 50% more worms than those which received cells or serum from untreated immune donors. DEC appears to cause suppression of functional immunity and worm expulsion is not histamine mediated.     

A two-fold effect ofL-1-tosylamide-2-phenylethyl chloromethyl ketone on the oxidative metabolism of guinea pig phagocytes

The protease inhibitor,L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), stimulated the O ₂ ^– production, H₂O₂ generation, oxygen consumption, and the hexose monophosphate shunt of guinea pig peritoneal polymorphs. Other protease inhibitors were not able to stimulate the metabolic burst of these cells. Maximum stimulation was obtained at 100 µM concentration of the compound. No stimulation was seen in human blood polymorphs even at concentrations higher than those effective on guinea pig polymorphs. TPCK also stimulated the oxidative metabolism of guinea pig blood polymorphs and of guinea pig resident peritoneal macrophages. At concentrations which did not stimulate the oxidative metabolism of guinea pig polymorphs, TPCK inhibited the O ₂ ^– production induced in these cells by treatment with phorbol myristate acetate (PMA) or with other soluble stimuli. Other protease inhibitors also inhibited the respiratory burst induced by PMA. It is concluded that TPCK exerts two effects on the metabolism of guinea pig phagocytes, which are probably mediated by different mechanisms. The inhibitory effect on the PMA-stimulated respiratory burst might be related to the antiprotease activity of TPCK, while the stimulation of the respiratory burst seems to be independent of protease inhibition.