Biomedical subjects
Publications and source records attributed to D A Stevens.
Two isolates of Candida dubliniensis were identified from a collection of 30 examined from Israel in a molecular epidemiology study. The 30 isolates were tentatively identified as Candida albicans. The new species, C. dubliniensis, is being reported from new geographic locales. These two isolates, from an Arab and a Druze patient, are the first to be reported from the Middle East.
The number of cases of systemic histoplasmosis has increased substantially in recent years, and improved therapy is needed. We examined the efficacy of immunomodulation with interferon (IFN)-gamma alone or in combination with a suboptimal regimen of amphotericin B for the treatment of primary systemic murine histoplasmosis. In the first study, BALB/c mice were infected with Histoplasma capsulatum G217B and treated with 10(5) U of IFN given every other day either preinfection and postinfection or only postinfection, alone or in combination with amphotericin B. IFN alone given subcutaneously (s.c.) postinfection prolonged survival over untreated controls (P < 0.01), whereas intravenous (i.v.) administration was ineffective. All combination regimens and amphotericin B alone significantly prolonged survival (P < 0.0001). The combination regimens of amphotericin B and IFN i.v. (pre- and postinfection) or IFN s.c. (postinfection) reduced the fungal burden in the liver and spleen; the latter regimen had superior efficacy in the spleen (P < 0.05) to either amphotericin B or IFN alone. After infection with a low-challenge inoculum, IFN given s.c. (pre- and postinfection) alone caused a significant reduction in fungal burden in the spleen (P < 0.001). In an acutely lethal model, combination regimens of IFN s.c. or i.v. and amphotericin B again prolonged survival (P < 0.01-0.001), with amphotericin B plus IFN given s.c. (pre- and postinfection) superior to all regimens (P < 0.05-0.01). This regimen also showed enhanced efficacy in causing the reduction of fungal burden in the spleen (P < 0.05). These results indicate that IFN in combination with AmB shows enhanced efficacy in the treatment of systemic histoplasmosis and support the potential utility of IFN as an adjunctive therapy.
GM-CSF is a potent stimulator of haematopoietic cells as well as some functions of granulocytes and macrophages. GM-CSF has many clinical uses; however, little is known about the effects of GM-CSF treatment in vivo on the responses of tissue lymphocytes in terms of secretion of Th-1 and Th-2 cytokines. We investigated this issue by measuring the responses of spleen cells from mice 24 h after treatment i.p. with saline or rmGM-CSF. GM-CSF at 16.7-50.0 microg/kg significantly increased (P < 0.01) spleen cellularity 2-2.5-fold and enhanced proliferative responses of non-stimulated (no mitogen) as well as concanavalin A (Con A)-stimulated spleen cells. Secretion of IFN-gamma by Con A (2.5 microg/ml)-stimulated spleen cells was significantly (P < 0.01) increased from 1.8 microg/ml by control spleen cells to 5.2 microg/ml by GM-CSF spleen cells. IL-10 production was greater (0.25 microg/ml, P < 0.05) by Con A-stimulated spleen cells from GM-CSF-treated mice compared to control spleen cells (0.06 microg/ml). By contrast, there were no significant differences in IL-4 production by Con A-stimulated spleen cells from the different groups. These results show that GM-CSF treatment increases spleen cellularity and primes lymphocytes for enhanced responses. The enhanced production of Th-1 cytokines by primed lymphocytes may partially explain the beneficial role of in vivo administration of GM-CSF in several clinical situations.
Host defense against systemic mycoses is multifactoral, depending on innate, as well as acquired, mechanisms. Innate resistance mechanisms include intact physical barriers, host proteins, nonspecific inflammatory responses, hormonal status, sex, and genetic make-up. However, the importance of any 1 factor in resistance to systemic fungal infections can vary depending on the causative agent. Macrophages and neutrophils play a critical role in the stasis or killing of these organisms by using the production of oxygen radicals, cationic proteins, nitric oxide (NO), and peroxides or iron deprivation. Although these cells are often ineffective in killing the organisms innately, activation of macrophages and neutrophils during an acquired immune response by the proinflammatory cytokine interferon-gamma as well as colony-stimulating factors increases the capacity of these cells for killing. A strong Th1 response can provide protective immunity, whereas a Th2 response can result in increased disease severity. The importance of native antibodies in resistance to mycoses remains in question.
Coccidioidal meningitis is lethal in the absence of treatment. The advent of the azoles has not led to cure, causing many clinicians to revert to intra-cerebrospinal fluid (CSF) amphotericin as part of the treatment regimen, desiring the influence of the latter regimen's ability to clear infection more completely and rapidly. Intra-CSF amphotericin therapy is, however, far more toxic than oral azoles and requires much more clinical management to achieve success and avoid toxicity. This management task increasingly, for insurance reasons or geographic reasons, falls on clinicians unfamiliar with the disease. We delineate our experience in the medical and surgical management of this form of therapy, including procedural details that we have found useful, for the benefit of our colleagues who may wish to use them. As ours is a teaching institution, we have found this material also useful for physicians in training, who are learning about the treatment of these patients.
The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.
The objectives of this study were to: (i) see if granulocyte macrophage colony-stimulating factor (GM-CSF) could protect bronchoalveolar macrophages (BAM) against suppression by dexamethasone (DEX) and (ii) test the combined effect of GM-CSF and DEX on lymphocyte responses. Murine BAM killed Aspergillus fumigatus conidia by 33 +/- 4% (mean +/- SD) in a 2.5-h assay, unaffected by GM-CSF treatment. Killing by BAM treated with DEX (10(-7) M) for 48 h in vitro was reduced to 13 +/- 6%; however, if GM-CSF (500 U ml(-1)) was present during DEX treatment of BAM, killing of conidia (33 +/- 2%) by BAM was preserved. By contrast, DEX suppression of lymphocyte responses to concanavalin A was maintained during co-culture with GM-CSF. In sequence treatment experiments, initial treatment of BAM with GM-CSF protected against subsequent treatment with DEX. When macrophages were pretreated with DEX, GM-CSF could reverse suppression even when added subsequently, provided DEX treatment was discontinued. These data suggest that it may be possible to suppress lymphocyte responses with DEX, yet at the same time maintain BAM defenses with GM-CSF against pulmonary infections by conidia of A. fumigatus.
The polyene partricin compound SPA-S-753 (Societa Prodotti Antibiotici, Milano, Italy) was assessed in a murine model of systemic candidosis. CD-1 mice were infected iv with Candida albicans and treated iv with SPA-S-753 or amphotericin B. All treatment regimens of SPA-S-753 or amphotericin B were equivalent and significantly prolonged survival compared with controls (P < 0.001). Amphotericin B and SPA-S-753 significantly reduced burdens of C. albicans in the spleen and kidneys. Overall, cure rates were similar, amphotericin B at 1 mg/kg cured three and SPA-S-753 at 10 mg/kg cured four mice of infection in both organs. The efficacy of SPA-S-753 is between equivalent and <10-fold as potent as amphotericin B. These results are encouraging and warrant further studies on SPA-S-753.
An earlier study reported that human cerebrospinal fluid (CSF) has fungistatic activity for Cryptococcus neoformans. The present study reports that molecular sieve fractionation of concentrated CSF yielded three protein peaks, one of which (p2) had anticryptococcal activity. On a DEAE-Sephacel anion-exchange column the active molecular sieve peak (p2) gave two peaks that contained anticryptococcal activity. The first (DEAE-1) eluted with 0.1 M NaCl and the second (DEAE-2) eluted with 0.2 M NaCl in buffer. Fungistatic activity of DEAE-1 was reversed by FeCl3. Moreover, FeCl3 reversed inhibition of C. neoformans growth by CSF. In contrast, activity of DEAE-2 was not reversed by FeCl3, indicating that inhibition was produced by an iron-independent mechanism. Immunoblot assays showed that transferrin was present in DEAE-1 but not in DEAE-2, whereas albumin was present in DEAE-2 but not in DEAE-1. On NuPAGE, DEAE-1 protein migrated as a single band corresponding to transferrin and DEAE-2 protein gave a single band corresponding to albumin. In control experiments, human serum albumin subjected to the same isolation protocol acquired anticryptococcal activity similar to that of DEAE-2. Therefore, CSF albumin (DEAE-2) activity was associated with the isolation protocol. These data indicate that transferrin, present in or isolated from CSF, sequesters trace amounts of ferric iron, inhibits growth of C. neoformans and acts as an innate defence mechanism.
A model of orogastric candidosis in SCID mice, which mimics disease seen in AIDS patients, was used to evaluate ravuconazole in comparison with fluconazole for treatment. Mice were infected orally with Candida albicans and received either no treatment or oral treatment once daily for 12 days with 1, 5, or 25 mg of ravuconazole per kg of body weight per day, 5 or 25 mg of fluconazole per kg per day, or diluent (10% dimethyl sulfoxide in 0.5% carboxymethyl cellulose). The numbers of C. albicans CFU in the esophagus, stomach, small intestine, and cecum on day 25 in mice given no treatment and diluent were equivalent. Both doses of fluconazole significantly reduced numbers of CFU in all four tissues but were equivalent to each other. Ravuconazole showed dose-responsive improvement of clearance of CFU. Ravuconazole at 25 mg/kg was superior in reduction of numbers of CFU in all tissues to controls or 25 mg of fluconazole per kg and to other regimens in at least three tissues. Fluconazole at 25 mg/kg cured no infection in any tissue, whereas 25 mg of ravuconazole/kg cleared infection in all tissues from 50% of mice. Ravuconazole has good efficacy and the potential to cure mucosal candidosis in the absence of a functional immune response.
The lipid formulation of amphotericin B, Amphotec (ABCD), has not been used intrathecally. After a single intrathecal dose or after four doses, conventionally formulated deoxycholate amphotericin B (AMB) (Fungizone) resulted in higher levels of amphotericin B in the cerebrospinal fluid of rabbits than did ABCD. Clinically and histologically, ABCD was about threefold less toxic than AMB after a single dose and 3- to 30-fold less toxic after multiple dosing. These data are encouraging for the potential use of ABCD as an intrathecal treatment.
We have previously shown that gamma interferon (IFN-gamma) is a useful adjunct to therapy of experimental systemic cryptococcosis in normal mice. To better emulate AIDS patients, SCID mice were infected intravenously with Cryptococcus neoformans. Mice received no therapy, 3 mg of amphotericin B (AmB) per kg of body weight, or 10(5) U of IFN-gamma alone (prophylactically and therapeutically or only therapeutically) or with AmB. In the first experiment, >75% of the mice survived. Therapy with AmB alone was efficacious compared to no therapy in all organs. Both regimens of IFN-gamma alone were efficacious in the brain and lungs, and the combination of AmB and IFN-gamma showed significant synergy in the kidneys. AmB alone cured 40% of mice of infection, whereas the combination regimens cured >50% of the mice and 90% of the brain infections. In a second study, IFN-gamma again proved efficacious alone, and when given with AmB its efficacy was improved. Therapeutic IFN-gamma alone was effective only in the liver compared to no therapy, and the combination regimen, although highly effective, showed no significant synergy. In a third experiment, AmB alone or in combination with IFN-gamma prolonged survival compared to no therapy or IFN-gamma alone. The combination regimen showed significant synergy over AmB alone in the brain, liver, kidneys, and lungs. AmB alone cured no mice of infections in more than two organs, whereas AmB in combination with IFN-gamma cured 55% of infections in three or more organs. These results indicate that IFN-gamma has therapeutic efficacy in severely immunodeficient animals, especially in combination with AmB. Significant synergistic activity was noted in all organs except the spleen. Overall, IFN-gamma has utility as an adjunctive therapy against systemic cryptococcosis in the severely immunocompromised host.
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Although no well-designed studies have been carried out, the available data support the use of corticosteroids for acute exacerbations of allergic bronchopulmonary aspergillosis (ABPA). Neither the optimal steroid dose nor the duration of therapy has been standardised but limited data suggest the starting dose should be prednisone (approximately 0.5 mg/kg/day). The decision to taper steroids should be made on an individual basis, depending on clinical course. The available data suggest that clinical symptoms alone are inadequate to make such decisions, since significant lung damage may occur in asymptomatic patients. Increasing serum IgE levels, new or worsening infiltrate on chest radiograph and worsening spirometry suggest that steroids should be used. Multiple asthmatic exacerbations in a patient with ABPA suggest that chronic steroid therapy should be used. Itraconazole appears useful as a steroid sparing agent.
Amphotericin B Hydrosomes (AH; Access Pharmaceuticals Inc) are a novel formulation of hydrophilic, heparin-surfaced nanoparticles (mean diameter 105 nm) containing amphotericin B (AmB) designed to target infected sites by local adhesion. AH are cleared in part by a hepatobiliary mechanism, which results in a reduction of AmB concentration in major organs by about 50% in 24 h. In mice with pulmonary blastomycosis, unlike Fungizone (Bristol-Myers Squibb Inc), a deoxycholate micellar formulation of AmB, AH accumulates 3-fold more in infected lungs than normal lungs, between 3 and 24 h post-injection. Histologically, AH accumulates at the sites of lesions. AH is approximately 7-fold less toxic than Fungizone based on acute lethality and histopathological assessment of renal damage. In vitro, AH and Fungizone were equally active against Blastomyces dermatitidis and in vivo they were equivalent in prolonging mouse survival, when compared with equal dosing of AmB. In reducing infectious burdens in vivo, Fungizone was 3-fold more effective than AH on a mg/kg basis of administered AmB. However, AH at 4.8 mg/kg cured 50 to 60% of mice, whereas Fungizone at a near lethal dose of 1.2 mg/kg cured none. The AH formulation of AmB has an improved therapeutic index, relative renal-site avoidance and selective accumulation in infected tissues, which combine to merit additional studies in appropriate fungal models.
Dexamethasone (DEX) is a potent immunosuppressive agent used in the treatment of several disorders. However, despite its beneficial effects, DEX puts patients at risk for opportunistic infections, especially pulmonary aspergillosis. Previously we reported that in vitro granulocyte-macrophage colony-stimulating factor (GM-CSF) blocks the immunosuppressive action of DEX on bronchoalveolar macrophages (BAMs). Here we report that BAMs freshly isolated from mice treated intraperitoneally with DEX for 24 h had significantly (P<0.01) reduced killing of conidia, i.e., 15 +/- 5% conidia killed by BAMs from DEX-treated mice versus 35 +/- 3% by BAMs from mice given saline, 38 +/- 5% by BAMs from mice given GM-CSF, and 39 +/- 1% by BAMs from mice given both DEX and GM-CSF. On the other hand, in another compartment GM-CSF could not block the DEX reduction of spleen weight and spleen cellularity. Unlike GM-CSF, granulocyte colony-stimulating factor did not block DEX suppression of BAMs. GM-CSF given 24 h before DEX resulted in blocking of DEX suppression of BAM conidiacidal activity. However, when DEX was given 24 h before GM-CSF, DEX suppression of BAM was not reversed. These data show that GM-CSF in vivo blocks the in vivo immunosuppressive effects of DEX on BAM killing of conidia and suggest a potential use of GM-CSF in patients at risk for aspergillosis due to immunosuppressive DEX treatment.
Aspergillomas are fungal balls within lung cavities. The natural history of patients affected is variable. Hemoptysis is a dangerous sequela. Factors associated with a poor prognosis have been defined and therapy is difficult because of the lack of a blood supply. Randomized trials are lacking. Surgical treatment is definitive but many patients are ineligible. Percutaneous therapy and bronchial artery embolization is appropriate for some patients and itraconazole has produced favorable results in several studies.
BACKGROUND: In previous open-label noncomparative clinical trials, both fluconazole and itraconazole were effective therapy for progressive forms of coccidioidomycosis. OBJECTIVE: To determine whether fluconazole or itraconazole is superior for treatment of nonmeningeal progressive coccidioidal infections. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: 7 treatment centers in California, Arizona, and Texas. PATIENTS: 198 patients with chronic pulmonary, soft tissue, or skeletal coccidioidal infections. INTERVENTION: Oral fluconazole, 400 mg/d, or itraconazole, 200 mg twice daily. MEASUREMENTS: After 4, 8, and 12 months, a predefined scoring system was used to assess severity of infection. Findings were compared with those at baseline. RESULTS: Overall, 50% of patients (47 of 94) and 63% of patients (61 of 97) responded to 8 months of treatment with fluconazole and itraconazole, respectively (difference, 13 percentage points [95% CI, -2 to 28 percentage points]; P = 0.08). Patients with skeletal infections responded twice as frequently to itraconazole as to fluconazole. By 12 months, 57% of patients had responded to fluconazole and 72% had responded to itraconazole (difference, 15 percentage points [CI, 0.003 to 30 percentage points]; P = 0.05). Soft tissue disease was associated with increased likelihood of response, as in previous studies. Azole drug was detected in serum specimens from all but 3 patients; however, drug concentrations were not helpful in predicting outcome. Relapse rates after discontinuation of therapy did not differ significantly between groups (28% after fluconazole treatment and 18% after itraconazole treatment). Both drugs were well tolerated. CONCLUSIONS: Neither fluconazole nor itraconazole showed statistically superior efficacy in nonmeningeal coccidioidomycosis, although there is a trend toward slightly greater efficacy with itraconazole at the doses studied.