Endotoxin-stimulated alveolar macrophage recruitment of neutrophils and modulation with exogenous surfactant

OBJECTIVE: To determine whether endotoxin-stimulated alveolar macrophages would attract neutrophils and whether exogenous surfactant treatment would modulate this chemoattraction. DESIGN: Alveolar macrophages were harvested from bronchoalveolar lavage fluid and neutrophils from the blood of anesthetized guinea pigs. SUBJECTS: Hartley guinea pigs. INTERVENTIONS: Alveolar macrophages were suspended in RPMI 1640 and stimulated with 1 microg/mL of lipopolysaccharide (LPS), the supernatant removed and the alveolar macrophages were incubated in either RPMI or RPMI with surfactant at two different doses (292 microg/mL or 875 microg/mL) for 16 hrs. MEASUREMENTS AND MAIN RESULTS: The supernatant was extracted from the alveolar macrophages and placed in a chemotaxis plate and the migration of neutrophils was measured. Chemotaxis of all cell types to be tested was measured by a change of absorbance on a microplate reader set at 492 nm. Results were compared with alveolar macrophages not stimulated with LPS, RPMI alone, and N formyl-methionyl-leucyl-phenylalanine (FMLP). The supernatant of the stimulated alveolar macrophages increased neutrophil chemotaxis as compared with unstimulated alveolar macrophages, and RPMI (p < .05). Surfactant treatment with 292 microg/mL significantly decreased LPS-stimulated alveolar macrophages induced neutrophil chemotaxis. Treatment with 875 microg/mL of surfactant did not alter neutrophil chemotaxis. CONCLUSIONS: Alveolar macrophages stimulation with LPS increased the chemotaxis of neutrophils. Treatment with surfactant at a concentration of 875 microg/mL did not alter neutrophil migration; however, treatment with 292 microg/mL significantly decreased neutrophil chemotaxis suggesting that at low concentrations, surfactant inhibits chemokine release and may reduce pulmonary neutrophil sequestration in vivo.     

Influence of methylprednisolone on the intracellular antimicrobial activity of erythromycin and clindamycin against Legionella pneumophila

We have investigated the effect of methylprednisolone on the intracellular activity of erythromycin and clindamycin in vitro. An assay system was developed for the determination of intracellular activity of antibiotics against Legionella pneumophila using guinea pig resident alveolar macrophages. Erythromycin at a concentration of 0.625 mg/L (5 x MIC) and clindamycin at a concentration of 8 mg/L (MIC) inhibited the growth of a single strain of L. pneumophila in macrophages, whilst ceftizoxime at a concentration of 0.625 mg/L (5 x MIC) did not. Methylprednisolone at therapeutic concentrations did not affect the intracellular antibacterial activity of either erythromycin or clindamycin against L. pneumophila. We found no direct effect of methylprednisolone on the intracellular antibacterial activity of either erythromycin or clindamycin.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.     

Experimental mixed infection with Yersinia enterocolitica and Listeria monocytogenes in guinea pigs

The pathogenesis and the cell immune response (CIR) of guinea pigs after mixed infection with Y. enterocolitica and L. monocytogenes was investigated. The guinea pigs were infected per os with 1.1 x 10(9) CFU Y. enterocolitica 0:3, (pYV+) and four days later with 1.1 x 10(9) CFU L. monocytogenes 4B. Clinical, paraclinical and morphological findings attending the infectious process were followed in dynamics up to the 28th day post infection (p.i.) with L. monocytogenes. The phagocyting activity of alveolar macrophages (aMa) was suppressed against Y. enterocolitica, in contrast to peritoneal macrophages (pMa) engulfing yersiniae more actively at the end of the study. Moreover, the tendency of augmented entering in both phagocytes of L. monocytogenes cells was well demonstrated, starting at the earlier intervals of examination. Histopathological studies showed a purulent meningoencephalitis and a catarrhal pneumonie, non-reactive micronecroses in the spleen and lymphadenitis catarrhalis in the mesenteric lymph nodes. Analysis of the T-cell immune response (T-CIR) showed maximal values in the spleen lymphocytes after Y. enterocolitica and L. monocytogenes mixed infection. The B-CIR occurred early (at the 7th day p.i.) and was maximal at the 28th day p.i. in blood lymphocytes. The results obtained demonstrated that the mixed infection of guinea pigs with Y. enterocolitica and L. monocytogenes runs has a non lethal, generalized illness with a dominant role of L. monocytogenes cells.     

Altered cytokine production and impaired antimycobacterial immunity in protein-malnourished guinea pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity

The hallmark of Legionnaires' disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.     

Superoxide dismutase activity in polymorphonuclear leukocytes and alveolar macrophages of protein malnourished rats and guinea pigs

Chronic protein insufficiency in utero was achieved by feeding to rat dams an 8% protein diet beginning before breeding and continuing until weaning. Thereafter, the in utero malnourished pups were fed the 8% protein diet. Superoxide dismutase (SOD), an enzyme which protects against superoxide radical, was quantitated in pellet and supernatant of polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM). The AM of rats malnourished in utero contained 1.7 times as much protein and 3 times as much total SOD activity as the AM of control rats. In PMN of these rats, the amount of protein and SOD activity was the same. Significantly larger number of Streptococcus mutans were located from the dental plaque of the in utero malnourished rats. AM isolated from female guinea pigs fed a 9% protein diet from weaning at 11 days of age contained 1.5 times as much protein and twice as much SOD activity as AM from normal guinea pigs. However their PMN contained smaller amounts of total protein and SOD activity. The malnourished guinea pigs had a mammary gland infection and a prolonged conjunctival infection following inoculation with Guinea Pig Inclusion Conjunctivitis indicating that these animals were more susceptible to these infections. The increase in total SOD activity and in the total amount of protein in AM suggests that chronic protein insufficiency leads to susceptibility to infection and to macrophage activation but has no similar effects on PMN.     

Comparison of amikacin dosing regimens in neutropenic guinea pigs with Escherichia coli infection

OBJECTIVE: To derive pharmacokinetic data for 3 amikacin dosing regimens in guinea pigs and to determine whether the antibacterial activity of 15 mg/kg of body weight given twice daily is equivalent to administering the drug more frequently. ANIMALS: 10 guinea pigs in pharmacokinetic trials, and 10 guinea pigs in pretreatment, control, and amikacin treatment groups. PROCEDURE: Amikacin pharmacokinetic data were determined in guinea pigs after single i.m. administration of 3.75, 7.5 and 15 mg/kg. Guinea pigs had been made neutropenic by treatment with cyclophosphamide. All guinea pigs were inoculated with 2.8 x 10(8) colony-forming units (CFU) of Escherichia coli in the thigh muscle, then were allotted to 5 groups: pretreatment (euthanized 4 hours after inoculation), control, and 3 amikacin treatment groups (3.75 mg/kg, q 3 h; 7.5 mg/kg, q 6 h; and 15 mg/kg, q 12 h). Amikacin administration was begun 4 hours after E coli inoculation and was continued for 72 hours. Numbers of E coli CFU in infected thigh muscle were determined for each guinea pig. RESULTS: Difference in survival between control and the amikacin-treated groups was significant. The E coli infection concentration (log10 CFU) increased significantly in the control, compared with the pretreatment, group. Infection concentration decreased significantly in all treatment groups, compared with the pretreatment group. There was no significant difference in bacterial killing among the 3 treatment groups. CONCLUSION: Amikacin had a significant effect on survival of neutropenic guinea pigs with E coli infection. Antibacterial activity did not differ among 3 doses of amikacin administered at different intervals. CLINICAL RELEVANCE: Aminoglycoside dosing regimen with high peak concentration and long drug-free interval is as efficacious as divided dose regimens.     

Effect of prenatal heat stress on brain growth and serial discrimination reversal learning in the guinea pig

Exposure of pregnant guinea pigs to an environmental temperature of 42 degrees C for 1 hr daily on Days 20-24 of gestation resulted in a significant reduction in the birthweight and brainweight of newborn offspring. These deficits persisted to early maturity and were still evident at 250 days of age following behavioral testing. Although some degree of postnatal neurogenesis and brain growth had occurred, this was not sufficient to compensate for the retarding influence of prenatal hyperthermia. Heat stress was also observed to seriously impair learning performance on the original discrimination task and this tendency persisted over the subsequent 4 reversals for both initial and perseverative errors. Impaired learning performance was related to reduction in brainweight. Animals with lower brainweights made significantly more initial, perseverative and total number of errors over the 5 problems. In addition, 12 of the 14 behaviorally tested stressed progeny had brainweights that were at least 2 standard deviations below the mean of the controls and as a result were classified as micrencephalic. Although heat stressed animals showed a significant reduction in the number of amacrine synapses in the inner plexiform layer of the retina, performance differences were not atrributed to changes in synaptic organisation of the retinal circuitry or to visual or other ophthalmic defects, but brain function. Animals with higher mean maternal poststressing core temperatures gave birth to progeny which had smaller whole and part wet-brainweights. Examination of the effect of poststressing core temperature on brainweight revealed that brain growth, independent of bodyweight, was retarded when this temperature elevated above 41.5 degrees C. This represented a rise of approximately 2.1 degrees C above normal and for each 1 degree C rise above this temperature, brainweight was reduced by 0.4227 g of the control value. Analysis of the atmospheric content of the incubator during stressing sessions ruled out the possibility that changes in level of oxygen and carbon dioxide may have contributed to the retarding influence on fetal development and learning ability in the guinea pig.     

Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.     

Rationale for total nephrectomy for suspected renal cell carcinoma

Effects of hyperthermia and cell densities on inhibitory activity of ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells were studied. When cells at a low density of 5 x 10(3)/ml were treated with 75 microM ascorbic acid for 1 h, DNA synthesis was inhibited after treatment at 37 degrees C and the inhibition was significantly enhanced at 42 degrees C. At a cell density as high as 1 x 10(5)/ml, however, inhibition did not occur at 37 degrees C or 42 degrees C. In contrast, dehydroascorbic acid was inactive even at a low cell density under similar conditions. Inhibitory effects of ascorbic acid on DNA synthesis were also markedly enhanced by treatment at 40 degrees C. DNA synthesis was not inhibited in the absence of the drug. Furthermore, mice transplanted with cells treated with a combination of 75 microM ascorbic acid and hyperthermia at 42 degrees C, considerably prolonged their survival time in comparison with untreated cells. Addition of ascorbic acid to hyperthermia is suggested to be an advantageous treatment for cancer.     

Inhibition of Salmonella typhimurium attachment to chicken cecal mucus by intestinal isolates of Enterobacteriaceae and lactobacilli

The ability of selected strains of Enterobacteriaceae or lactobacilli isolated from the intestines of adult chickens to inhibit in vitro attachment of Salmonella typhimurium 3333/O to cecal mucus in the presence or absence of D-mannose was determined. Attachment in the absence of mannose was reduced by prior exposure of mucus to cultures of two isolates of Enterobacteriaceae, an Escherichia coli and a Hafnia alvei strain, but not to a third isolate, an Enterobacter agglomerans strain. Attachment of S. typhimurium was not inhibited when mannose was present in the blocking or attachment step. Formation of fimbriae by the two inhibitory Enterobacteriaceae strains and the S. typhimurium strain, as indicated by titers of mannose-sensitive hemagglutination of guinea pig erythrocytes was optimal in Z biphasic medium (consisting of tryptone, yeast extract, dextrose, and NaCl) incubated anaerobically at 42 C. Fimbriae of each of three strains prepared from these cultures also inhibited attachment. These are characteristics consistent with attachment and inhibition of attachment mediated by a mannose-sensitive adhesin associated with type 1 fimbriae on bacterial cells of Enterobacteriaceae strains. Attachment in the presence of mannose was significantly reduced by prior exposure of mucus to cultures of a Lactobacillus salivarius strain and a Lactobacillus delbrueckii delbrueckii strain but not to a strain of Lactobacillus for which the species had not been determined. Washed cells or spent culture supernatant fluid from brain-heart infusion broth, Z broth, or Z biphasic cultures of the inhibitory strains of lactobacilli incubated at 37 or 42 C inhibited this form of attachment. Of 27 intestinal isolates of Enterobacteriaceae and 21 of lactobacilli, the lactobacilli strains were generally more hydrophobic than the Enterobacteriaceae as determined by adherence to hexadecane. The lactobacilli isolates did not agglutinate guinea pig erythrocytes. The data suggest more than one mechanism for mediating attachment of inhibitory bacterial strains and for subsequent attachment of S. typhimurium.     

Assessment of the expression of p53, MIB-1 (Ki-67 antigen), and argyrophilic nucleolar organizer regions in carcinoma of the extrahepatic bile duct

Group B streptococci (GBS) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS-macrophage interaction in the absence of type-specific antibodies, we examined the features of GBS survival in thioglycollate-elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC)-dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS-Ia) and type III GBS strain COH 31r/s (GBS-III), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon-gamma (IFN-gamma) and lipopolysaccharide from Escherichia coli (LPS) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN-gamma and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro-organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12-myristate 13-acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBS-infected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC-dependent cell signalling was also demonstrated by the reduced c-fos gene expression in GBS-infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival.     

Production of chemoattractant by Helicobacter pylori

Helicobacter pylori is present in the antral region of the stomach in a majority of patients with gastritis type B. The specific mechanism whereby the organism participates in the development of disease remains uncertain. Since the organism is not invasive, we postulate that H. pylori produces a chemoattractant that recruits inflammatory cells to the antral region of the stomach. H. pylori was grown under microaerophilic conditions at 37 degrees C for 72 hr in Brucella broth containing 1% fetal bovine serum. Culture supernates were harvested after removal of organisms by centrifugation and filtration. The putative chemoattractant in culture supernates as well as that which might be present endogenously in the growth medium (negative control) was assayed against human neutrophils (PMN) in modified Boyden blind-well chambers using 3.0-microns membranes. We found that H. pylori supernates are chemotactic and showed up to 130% activity when compared to the positive chemoattractant control (zymosan-activated serum, a source of C5a). Minimal activity was observed with virgin growth medium. The chemoattractant activity is proportional to the number of colony forming units (CFU) of H. pylori. Preliminary characterization of the activity shows that the chemoattractant is stable in a boiling water bath for 15 min, activity is lost within 1 hr in acid or alkali, and the chemotactic factor has an approximate molecular weight of 8500 daltons. The factor has no amino-sugar and is negative for the lipid A portion of lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.     

Enhancement by hyperthermia of the in vitro cytotoxicity of mitomycin C toward hypoxic tumor cells

Mitomycin C and hyperthermia are both toxic to chronically hypoxic EMT6 tumor cells. Combinations of this drug and heat were tested in vitro in normally aerated and chronically hypoxic EMT6 mouse mammary tumor cells to establish whether greater than additive cytotoxicity could be achieved by combined treatment. Cell survival was measured at four concentrations of mitomycin C (0.01, 0.1, 1.0, and 10 microM) at 37 degrees or at elevated temperatures (41, 42, and 43 degrees) for durations of 1, 2, 3, and 6 hr. At 42 degrees, exposure to mitomycin C for 3 and 6 hr produced a 2- to 3-fold increase in hypoxic tumor cell kill at all drug concentrations over that expected for strict additivity. A 15-fold enhancement in the kill of hypoxic tumor cells was obtained at 1.0 and 10 microM mitomycin C at 43 degrees for 6 hr of exposure. Under most conditions, additivity was observed for the antibiotic and heat in oxygenated cells, except at 43 degrees with 0.01 and 0.1 microM mitomycin C following 3 and 6 hr of treatment, conditions under which a 5- to 10-fold potentiation of tumor cell kill was obtained. The rate of formation of reactive metabolites from mitomycin C under anaerobic conditions in EMT6 cell-free preparations was measured. A 30 to 50% increase in alkylating activity was observed at elevated temperatures, suggesting that the enhanced cytotoxicity of mitomycin C with heat toward hypoxic cells may, in part, be due to an increase in activation of the drug.     

Suppression of the granulocyte colony-stimulating factor response to Escherichia coli challenge by alcohol intoxication

Alcohol's suppressive effects on polymorphonuclear leukocyte (PMN) production and function increases host susceptibility to a wide variety of infections and impairs the ability of these effector cells to seek and destroy invading pathogens. Granulocyte colony-stimulating factor (G-CSF), an important regulator of PMN production and function, is known to be increased in the plasma during infectious episodes. In previous studies we found acute alcohol intoxication to suppress the tumor necrosis factor-alpha (TNF alpha) response to in vivo challenges with bacteria or lipopolysaccharide. The present study was initiated to determine the impact of alcohol intoxication on the plasma G-CSF response to gram-negative infection. For this purpose, rats received an intravenous challenge of Escherichia coli (10(6) CFU) 30 min after an intraperitoneal injection of ethanol (5.5 g/kg) or an equivalent volume of saline (control). Ethanol-intoxicated rats had a greater 48 hr mortality to live E. coli injection than did unintoxicated animals (45% vs. 8%). Despite an increased bacterial burden in both the lung and liver at 24 hr after initiating E. coli infection in alcohol-intoxicated animals, PMN tissue recruitment, indexed as myeloperoxidase activity, did not differ between control and alcohol-treated rats. Moreover, alcohol suppressed blood PMN phagocytic capacity to a greater extent in animals given alcohol than controls at 5 and 24 hr after initiating infection. In control animals after intravenous E. coli injection, bioactive G-CSF increased in plasma and peaked near 300 ng/ml at 8 hr. In rats pretreated with alcohol, the plasma G-CSF response was markedly suppressed in response to intravenous E. coli (p < 0.05). In a second experiment, neutralization of the E. coli-induced plasma TNF alpha response by pretreatment with anti-TNF alpha antibody similarly inhibited the plasma G-CSF response. These results support the postulate that alcohol-induced inhibition of TNF alpha directly contributes to the adverse effects of alcohol on PMN function by suppressing the normal autocrine amplification pathway responsible for G-CSF production.     

Antagonistic effect of human alpha-1-antitrypsin on excystation of Cryptosporidium parvum oocysts

This study evaluated the effects of the human serine protease inhibitor alpha-1-antitrypsin (AAT) on in vitro excystation and infectivity of Cryptosporidium parvum. Excystation was monitored at 37 C in RPMI medium in the presence of 0, 100, 500, or 1,000 micrograms/ml AAT. AAT significantly inhibited (P < 0.05) excystation of bleach-decontaminated oocysts in a concentration-dependent manner at incubation intervals from 15 to 90 min but did not alter the excystation dynamics of unbleached oocysts. Bleach-treated oocysts, suspended in RPMI containing 0, 1, 10, 100, 500, or 1,000 micrograms/ml AAT, were used to inoculate bovine fallopian tube epithelial (BFTE) cell monolayers. Alternately, sporozoites, excysted at 37 degrees C and collected by filtration, were used to inoculate BFTE cells under the same conditions. The mean number of parasites counted in AAT-treated, oocyst-inoculated cells was significantly less (P < 0.01) than control mean values at 24 and 48 hr post-inoculation (PI); longer PI intervals (72-96 hr) exhibited a decreased inhibitory effect. AAT did not inhibit parasite infection when cultures were inoculated with C. parvum sporozoites. The findings of this study show that the anticryptosporidial potential of AAT is primarily associated with an antagonistic effect on oocyst excystation.