Pimozide blocks establishment but not expression of amphetamine-produced environment-specific conditioning.

Animals with a history of receiving daily injections of +-amphetamine in a specific environment showed a placebo effect (enhanced activity) when injected with saline and placed there; control animals with similar but dissociated drug histories and experience with the test chamber failed to show the effect. The dopamine receptor blocker pimozide antagonized the establishment of conditioning. However, the same dose of pimozide, when given to previously conditioned animals on the placebo test day, failed to antagonize the expression of conditioned activity. Thus, during conditioning dopaminergic neurons mediated a change that subsequently influenced behavior even when dopaminergic systems were blocked. Although schizophrenia may be related to hyperfunctioning of dopamine, neuroleptic drugs, which block dopamine receptors on their first administration, do not have therapeutic effects for a number of days. The results of the pimozide experiments may resolve this paradox.

Characteristics of dermal invasion in experimental cutaneous candidiasis of leucopenic mice.

The course of experimental cutaneous Candida albicans infections produced in mice made leucopenic by the administration of cyclophosphamide was compared to that in untreated animals. In the latter, neutrophils characteristically infiltrated the area of infection and the organisms were virtually always confined to the epidermis. However, even though many fewer foci of infection were associated with neutrophils in the cyclophosphamide-treated animals, a majority of these foci were also unable to penetrate past the epidermis. Although Candida yeast proliferated relatively poorly when cultured in homogenates of skin lacking the epidermis, Candida pseudohyphae could invade into the dermis if inoculated skin was isolated from normal animals and cultured in vitro, or if the epidermis was removed by gentle scraping prior to inoculation with Candida yeast onto the remaining skin of leucopenic animals. Therefore, in the absence of neutrophil contact and killing of Candida pseudohyphae in the epidermis, other cutaneous defense mechanisms appear to be capable of preventing invasion of a majority of the organisms into the dermis. These findings may help to explain why deep Candida infections are rare in patients who have extensive superficial candidiasis.

Effects of extinction, pimozide, SCH 23390, and metoclopramide on food-rewarded operant responding of rats.

The similarity in the pattern of responding produced by extinction and dopamine (DA) receptor blockers has led to the suggestion that DA neurons may participate in the usual effects of reward on behaviour. The purpose of the present study was to evaluate the effect of receptor-subtype specific DA antagonists on food-rewarded operant responding. Rats were trained to lever press for food on a variable interval 30-s schedule. They then received one of the following treatments prior to testing on the next 5 days: saline, nonreinforcement, the DA receptor blocker pimozide (0.5 or 1.0 mg/kg), the D1 receptor blocker SCH 23390 (0.01, 0.05, 0.1 mg/kg), and the D2 receptor blocker metoclopramide (1.0, 5.0, 10.0 mg/kg). Nonreinforcement resulted in both intra- and intersession declines in responding. The drugs produced dose-dependent decreases in overall responding. Additionally, both doses of pimozide and the higher doses of SCH 23390 and metoclopramide altered intrasession patterns of responding when compared to saline, with their greatest effect being in the latter portion of the session. Intersession declines were seen with the highest doses of SCH 23390 and metoclopramide and control studies showed that these declines could not be attributed to a buildup of the drug with repeated dosing. It was concluded that both D1 and D2 receptors participate in the control of behaviour by reward.

Epidermal proliferation and the neutrophilic infiltrates of experimental cutaneous candidiasis in mice.

A mouse model of cutaneous candidiasis was used to determine if the prominent neutrophilic infiltrates in the infected skin of nonimmune animals were responsible for inducing the early phase of epidermal proliferation seen in these infections. Both the organisms and resulting neutrophilic microabscesses were found in the cellular layers of the epidermis at 12 h after inoculation, and were then extruded together to a more superficial site in the stratum corneum over the next 1-2 days. The degree of epidermal proliferation elicited at the site of the Candida foci, as determined from the thickness of the cellular layers of the epidermis, was the same for foci with neutrophils as for those without, even when the latter came from severely leukopenic animals. The location of neutrophils within the infected skin or the numbers of organisms present did not seem to make a difference with respect to the degree of epidermal proliferation produced at the site of Candida foci. These data suggest that in acute experimental cutaneous Candida infections the organisms can elicit a vigorous epidermal proliferative response in the absence of the neutrophilic infiltrates usually seen in these infections.

Use of a microtiter plate assay to detect the rate of killing of adherent Candida albicans by antifungal agents.

OBJECTIVES: Candida albicans may become adherent to prosthetic devices of various kinds and thereby produce infections that are difficult to treat with standard antifungal therapy. The objective of the present work was to study the effectiveness of antifungal agents against adherent C. albicans yeast cells. STUDY DESIGN: A microtiter plate assay was developed to assess the time required for killing of the fungal cells by three antifungal agents. RESULTS: The assay initially was validated by demonstrating that the percentage of organisms adhering to the test wells was relatively constant and that exposure to the antifungal agents caused only minimal dislodgement of viable organisms from the plates. In studies that used this assay to determine the time required for killing the adherent yeast cells, chlorhexidine was found to be the most effective; in fact, in comparing the minimal lethal concentrations of the agents for exposures of 2 minutes versus 4 hours, a ratio of 2.9 was obtained for chlorhexidine versus 1050 for amphotericin B and 556 for nystatin. CONCLUSION: The microtiter plate assay used in these studies may therefore be useful as a screening test to determine which antifungal agents have the most rapid fungicidal effects on adherent fungal organisms.

Effect of zinc-reversible growth-inhibitory activity in human empyema fluid on antibiotic microbicidal activity.

Abscess fluid supernatants have zinc-reversible microbial growth-inhibitory activity that is mediated by calprotectin, a zinc-binding protein. Because it inhibits microbial growth, this activity might interfere with killing by antibiotics that require their target organisms to be proliferating. In the present study, we cultured bacteria in human empyema fluid and used zinc to overcome the growth-inhibitory effect of calprotectin. We then compared the effect of zinc on killing by the beta-lactams ampicillin and cefazolin with that of the fluoroquinolone trovafloxacin, since the latter may be better able to kill nonproliferating organisms. In empyema fluid diluted 1:5 in normal saline, addition of zinc (30 microM) increased growth of two strains of Staphyloccocus aureus and two strains of Escherichia coli but did not affect the MICs or MBCs of the three antibiotics in Mueller-Hinton broth. For one strain of S. aureus, no effect of zinc was found on killing by either ampicillin or cefazolin. However, with the other strain of S. aureus and both strains of E. coli, significant enhancement of killing by both drugs was observed with zinc addition. On the other hand, no effect on the killing of any of the organisms was observed for trovafloxacin when zinc was added. These results suggest that the zinc-reversible growth-inhibitory activity of abscess fluid may interfere with the microbicidal activity of antibiotics requiring proliferating target organisms, although antibiotics better able to kill nonproliferating organisms may be less affected by this phenomenon.

Effect of immunosuppression on epidermal defenses in a murine model of cutaneous candidiasis.

A number of pharmacologic treatment regimens were used to evaluate the early defenses against experimental cutaneous candidiasis in nonimmune mice. Severe immunosuppression of the animals was found to have little effect on the numbers of Candida pseudohyphae that initially infected the skin, but it did, however, modify the progression of the infection afterwards. Of the regimens tested, only the combination of intraperitoneal cyclophosphamide and intravenous phorbol myristate acetate both completely eliminated the epidermal neutrophilic infiltrates characteristic of these infections and promoted a significant degree of Candida invasion into the dermis. However, the epidermal proliferative response to the infections, generally considered to be an important mechanism of defense against superficial mycoses, was equivalent at the sites of both invasive and noninvasive foci, and it was generally comparable to that in normal animals. Dermal invasion in the treated animals was also found to occur at a time (between 12 and 24 hours after inoculation) when the epidermis was maximally proliferating. In contrast to these results, the intraperitoneal administration of colchicine significantly suppressed epidermal proliferation at the Candida foci but had only minimal effects in promoting dermal invasion. Therefore, whereas epidermal proliferation could be involved in the eventual clearance of these experimental cutaneous Candida infections, this mechanism apparently has little to do with either limiting the number of organisms initially infecting the skin or preventing their invasion into the dermis.

Inhibition of pseudohyphal growth as a neutrophil-mediated host defense mechanism against experimental deep Candida albicans infections in mice.

Neutrophils have recently been found to have microbistatic activity in addition to their well-described microbicidal mechanisms; therefore, a murine model of deep candidiasis was used to evaluate the possibility that both antimicrobial activities might participate in the host defense against this type of infection. In nonleukopenic animals, subcutaneous injection of Candida albicans yeast cells produced local abscesses and early metastatic spread of the infection to the kidneys. Although completely isolated within a dense neutrophilic infiltrate, a significant proportion of the fungal cells in the subcutaneous abscesses of these animals remained viable for at least 6 to 10 days after inoculation; the infections at this site were observed to resolve after spontaneous rupture of the abscesses. Growth of the organisms appeared to be suppressed at later time points in these animals, as evidenced by the markedly reduced proportions of multicelled pseudohyphae (representing organisms that had undergone cell division) observed in their abscess exudates as compared with those in samples from leukopenic animals. In addition, at 10 days after inoculation, very few multicelled pseudohyphae were observed histologically in the subcutaneous infections of nonleukopenic animals, whereas masses of these forms were found in leukopenic ones. Fluids from the abscesses of the nonleukopenic animals appeared to contain growth-inhibiting activity for C. albicans, in that the organisms could not grow in them unless additional zinc were to be added to the medium. This type of zinc-reversibility is a characteristic of neutrophil microbistatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate of individual organisms during clearance of experimental cutaneous Candida albicans infections in mice.

A mouse model of acute cutaneous Candida albicans infections was used to study the manner in which these infections are cleared. Results of histological examination were correlated with determinations of the viability by acridine orange staining of superficial C. albicans pseudohyphae retrieved from the surface of the infected skin. The number of organisms retrieved from the skin surface was highest on the third and fourth day after inoculation, a finding which appeared to relate to a loss of Candida foci observed histologically to occur after the second day. Viability was high (approximately 80%) for at least 1-2 days after the organisms were seen histologically to have become associated with neutrophils and extruded from the stratum Malpighi into the stratum corneum; however, at later time points (fourth and fifth day after inoculation), the viability of the retrieved organisms did decline. Pseudohyphae germinated in vitro and applied to the skin of mice were found to be non-viable when retrieved 24 h later. These data suggest that the microbicidal processes of neutrophils may not be required for resolution of these infections. They are most consistent with clearance through an epidermal proliferative response which relocates the infecting organisms to a very superficial site, from which they can be either lost in a viable state, or subjected to killing by other factors at the skin surface.

Arrays of Candida albicans pseudohyphae that protect the organisms from neutrophil fungicidal mechanisms in experimental infections of mice.

Experimental subcutaneous Candida albicans infections in mice were used to examine the manner in which this pathogen is cleared in animals recovering from cyclophosphamide-induced leucopenia. In this system, infections at the inoculation sites progressed rapidly during a 6 day period of leucopenia to form arrays of parallel filamentous organisms that effectively isolated those in the interior from contact by neutrophils, even when the leucopenia had resolved. Dense collections of organisms also developed at sites of metastatic infection in the kidneys. A majority of the organisms were found to be viable when they were retrieved from the infected subcutaneous sites of animals that had recovered from leucopenia and whose abscesses had begun to drain spontaneously. Removal of the protective arrays of fungal cells appeared to be accomplished by drainage of abscess contents through the surface of the skin or into the collecting system of the kidney. Drainage of the subcutaneous abscesses did not occur in the cyclophosphamide-treated animals until after the neutrophilic infiltrates had developed, suggesting that this drainage process was mediated by neutrophils rather than by the organisms themselves. In summary, the above findings demonstrate that C. albicans infections in leucopenic hosts may progress to the extent that they would be very difficult to clear solely through the microbicidal processes of returning neutrophils. However, neutrophils also appear to promote the removal of masses of viable fungal cells to the exterior of the body.

Inhibition of Candida albicans growth by calprotectin in the absence of direct contact with the organisms.

Calprotectin is a calcium- and zinc-binding protein that is present in neutrophil cytoplasm and abscess fluid supernatants. This protein appears to inhibit microbial growth through competition for zinc; however, experiments to show that calprotectin can inhibit growth of microorganisms across filter membranes have yielded conflicting results to date. To prevent recontamination of the filtrate by zinc in this type of experiment, Candida albicans was cultured on filter membranes placed on top of an agarose gel containing calprotectin. In these studies, calprotectin in the gels underneath did suppress growth on top of the filters, an effect reversible by 30 microM ZnSO4. In other experiments, the protein did not adhere to the organisms and later suppress their growth. These results indicate that calprotectin inhibits C. albicans growth in the absence of direct contact with the organisms; the findings support a zinc-deprivation mechanism of antimicrobial activity for this protein.

Effect of metals on Candida albicans growth in the presence of chemical chelators and human abscess fluid.

Calprotectin is a calcium- and zinc-binding protein that is present in abscess fluid supernatants and appears to inhibit microbial growth through competition for zinc. In the present study, growth inhibition by chemical chelators was compared with that produced by human abscess fluid to determine whether other chelators, perhaps with different metal specificities, would have the same or different patterns of metal reversibility as abscess fluid. Zinc was found to be more potent than the other metals tested in reversing C. albicans growth inhibition by human abscess fluid and three chemical chelators, even though in some cases the stability constants of certain of these chelators were higher for other metals. For example, in the presence of the chelator diethylenetriaminopentaacetic acid, zinc stimulated Candida growth at a 10-fold lower concentration than did iron, even though this chelator has a stability constant for iron that is almost 10(10) higher than that for zinc. These results suggest that the zinc specificity of calprotectin's C. albicans growth inhibition can best be explained by the marked sensitivity of this organism to zinc deprivation rather than by selective binding of this metal by the protein.

Resistance of zinc-supplemented Candida albicans cells to the growth inhibitory effect of calprotectin.

Calprotectin is a neutrophil cytoplasmic protein with significant microbistatic activity. This protein may compete for zinc, or the metal may inactivate a different microbistatic activity of the protein. To distinguish between these possibilities, the sensitivity to calprotectin was determined for zinc-supplemented Candida albicans cells. The latter had increased growth in cultures containing either human empyema fluid as a source of calprotectin or purified calprotectin itself. This increased growth did not appear to be due to leakage of zinc into the medium. In other experiments, empyema fluid supernatants did not suppress C. albicans growth across a dialysis membrane; however, other studies suggested that it is difficult to significantly suppress C. albicans growth by zinc depletion unless the depleting agent remains in the cultures. These results indicate that calprotectin inhibits C. albicans growth through competition for zinc.

Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.

Inefficient delivery of yeast cells as an explanation for reduced plating efficiency of Candida albicans.

The plating efficiency for fungal yeast cells is usually less than that expected from microscopic counts, and a number of explanations for this phenomenon have been proposed. The present study was undertaken to explore possible reasons for reduced plating efficiency of Candida albicans. Explanations that we evaluated and found unlikely included: ineffectiveness of different culture media and/or incubation temperatures for growing colonies, insufficient area of the plate available for expression of individual colonies, production of microcolonies, and inaccurate counting of the organisms in the inoculum. An assay for delivery of the inoculum into tissue culture plate wells indicated that reduced delivery of the organisms accounted for lower than expected plating efficiency. C. albicans yeast cells grown under low glucose conditions and expected to have reduced adhesiveness were found to have higher values for both delivery and plating efficiency in our assays. In summary, our results indicate that reduced plating efficiency for C. albicans under the conditions used for these experiments is best explained by the loss of some yeast cells during preparation of the inocula or delivery of the yeast cells onto the plates.

Effect of abscess fluid supernatants on the kinetics of Candida albicans growth.

Abscess fluid supernatants have been found to inhibit microbial growth, an effect that appears to be due to a protein originating in the cytoplasm of neutrophils. The antimicrobial activity of calprotectin, the responsible protein, is reversible by the addition of zinc to the culture medium. To more carefully analyze this type of antimicrobial activity supernatants of fluids from experimental subcutaneous Candida albicans abscesses in mice were studied to determine how they affect the in vitro growth kinetics of C. albicans. The abscess fluid supernatants inhibited growth in a dose-dependent fashion and more at early times than at later ones. Instability of the abscess fluid antimicrobial activity did not appear to explain the timing of the growth inhibition. A marked lengthening of the lag phase of growth was observed in cultures containing the supernatants (from approximately 6 hr in the control cultures to 15-20 hr in those with the supernatants). On the other hand, the abscess fluid supernatants had only minimal effects on the generation times of actively proliferating C. albicans yeast cells. In addition, these supernatants did not appear to significantly affect the percentage of inoculated organisms undergoing cell division, as determined by a limiting dilution assay. Therefore, these results indicate that abscess fluid supernatants extend the lag phase of C. albicans growth, an effect similar to that seen with zinc-deprived organisms.

Inefficiency of in vivo candidacidal mechanisms in experimental subcutaneous infections with Candida albicans in mice.

A murine model of subcutaneous Candida albicans infections was used to evaluate host defenses against inocula of from 10(1) to 10(8) yeast cells. In these experiments, small inocula did not produce abscesses that drained to the skin surface, whereas larger ones did. Also, small numbers of organisms often remained at the infected sites for up to 21 days after inoculation with either small or large numbers of organisms. The data from these studies suggest that the in vivo candidacidal mechanisms in these infections are relatively inefficient and that they therefore may require some additional mechanism to control proliferation of the remaining organisms.

Comparison of the metal-binding anticandidal activities of serum and abscess fluid supernatants.

Serum transferrin appears to play a role in host defense by competing with invading microorganisms for iron. The purpose of the present study was to compare this activity to a similar one recently described in abscess fluids and based on a calcium- and zinc-binding protein called calprotectin. Serum and abscess fluid supernatants were collected and pooled from groups of five to 10 C57BL/6 mice with experimental Candida albicans abscesses; serum was also collected from normal animals. In four experiments, serum was found to reduce in vitro C. albicans growth in Sabouraud glucose broth by a mean of 97.9% at 10 mg ml-1 of protein; this effect was reversed by adding 3-10 microM FeCl3, but not by similar amounts of ZnSO4. Abscess fluid supernatants had a greater effect, reducing growth by 99.9% at 1 mg ml-1 and 76.1% at 0.1 mg ml-1 of total protein; this effect was reversed by 3-10 microM ZnSO4, but not FeCl3. Although abscess fluid supernatants were effective when high inocula (10,000 yeast cells) were used, serum from the infected mice inhibited growth only with lower inocula (10-100 yeast cells). In a separate study, serum from infected mice (eight pools) reduced growth (by a range of 36 to 97%), whereas serum from normal mice (five pools) actually enhanced growth in this system (by a range of 173 to 595%).(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial activity of calprotectin isolated from human empyema fluid supernatants.

Abscess and empyema fluid supernatants have zinc-reversible antimicrobial activity that is thought to be due to calprotectin, a calcium- and zinc-binding protein present within neutrophil cytoplasm. The present studies were undertaken to determine if calprotectin isolated from human empyema fluid supernatants demonstrated similar antimicrobial activity to that of the original specimens. The characteristics of the calprotectin complex on SDS-PAGE and Western blotting with specific antisera were similar in neutrophil lysates and in empyema fluid supernatants. Ion-exchange and size-exclusion chromatography were used to obtain highly purified preparations of calprotectin from empyema fluids, and these preparations demonstrated zinc-reversible anti-Candida albicans activity which was similar to that observed in the original specimens. These findings suggest that calprotectin is responsible for most of the growth-inhibitory activity of empyema fluid supernatants against this organism.

Analysis of fluconazole effect on Candida albicans viability during extended incubations.

Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. However, recent work has demonstrated that this drug can reduce Candida albicans viability during prolonged incubations under non-growing conditions. The present study was undertaken to examine more closely some of the parameters of this killing activity. Fungicidal effects of 1.0 microg ml-1 of fluconazole were found during 7-14-day exposures in each of two media that prevented proliferation, distilled water and metal-depleted RPMI 1640 tissue-culture medium. Fluconazole appeared to be stable after being incubated at 37 degreesC for either 7 or 14 days. Strains of C. albicans resistant to fluconazole in standard short-term growth-inhibition assays were also found to be resistant to fluconazole's effect on viability in prolonged culture, suggesting similar mechanisms of action for these effects. C. albicans yeast cells pre-incubated for 7 days in distilled water were not more sensitive to the drug in short-term susceptibility assays. Although all proliferation of the organisms in distilled water cultures appeared to cease after 3 days, fluconazole added at 7 days still reduced C. albicans viability. Therefore, the drug appeared to kill the non-proliferating organisms directly rather than preventing growth and thereby the emergence of younger organisms that would live longer. Transmission electron microscopy demonstrated damage to the cell wall-cell membrane complex and interior contents of yeast cells incubated in distilled water alone; fluconazole appeared to increase the percentages of cells so affected. In summary, extended-incubation susceptibility tests demonstrated that fluconazole has direct fungicidal activity of non-proliferating C. albicans yeast cells. These results may be relevant to the manner in which this drug promotes clearance of chronic fungal infections.