Meta-analysis of spineless cactus feeding to meat lambs: performance and development of mathematical models to predict dry matter intake and average daily gain.

Spineless cactus is a useful feed for various animal species in arid and semiarid regions due to its adaptability to dry and harsh soil, high efficiency of water use and carbohydrates storage. This meta-analysis was carried out to assess the effect of spineless cactus on animal performance, and develop and evaluate equations to predict dry matter intake (DMI) and average daily gain (ADG) in meat lambs. Equations for predicting DMI and ADG as a function of animal and diet characteristics were developed using data from eight experiments. The dataset was comprised of 40 treatment means from 289 meat lambs, in which cactus was included from 0 to 75% of the diet dry matter (DM). Accuracy and precision were evaluated by cross-validation using the mean square error of prediction (MSEP), which was decomposed into mean bias, systematic bias and random error; concordance correlation coefficient, which was decomposed into accuracy (Cb) and precision (ρ); and coefficient of determination (R2). In addition, the data set was used to evaluate the predicting accuracy and precision of the main lamb feeding systems (Agricultural and Food Research Council, Small Ruminant Nutritional System, National Research Council and Institut National de la Recherche Agronomique) and also two Brazilian studies. The DMI, CP intake (CPI), metabolizable energy (ME) intake and ADG increased when cactus was included up to 499 g/kg DM (P<0.001). In contrast, animals fed high levels of cactus (>500 g/kg DM) had a decreased DMI, CPI and NDF intake, but increased feed efficiency (P<0.001) and similar ADG compared with those without cactus addition. The DMI was positively correlated with initial BW, final BW, concentrate and ADG, while it was negatively correlated with cactus inclusion and ME of the diet. On other hand, ADG was positively correlated with DMI, initial and mean BW and concentrate, and it was negatively correlated with cactus inclusion. The two developed equations had high accuracy (Cb of 0.95 for DMI and 0.94 for ADG) and the random error of MSEP was 99% for both equations. The precision of both equations was moderate, with R2 values of 0.53 and 0.50 and ρ values of 0.73 and 0.71 for DMI and ADG, respectively. In conclusion, the developed equation to predict DMI had moderate precision and high accuracy, nonetheless, it was more efficient than those reported in the literature. The proposed equations can be a useful alternative to estimate intake and performance of lambs fed cactus.

Invited review: Current production trends, farm structures, and economics of the dairy sheep and goat sectors.

Dairy small ruminants account for approximately 21% of all sheep and goats in the world, produce around 3.5% of the world's milk, and are mainly located in subtropical-temperate areas of Asia, Europe, and Africa. Dairy sheep are concentrated around the Mediterranean and Black Sea regions, where their dairy products are typical ingredients of the human diet. Dairy goats are concentrated in low-income, food-deficit countries of the Indian subcontinent, where their products are a key food source, but are also present in high-income, technologically developed countries. This review evaluates the status of the dairy sheep and goat sectors in the world, with special focus on the commercially and technically developed industries in France, Greece, Italy, and Spain (FGIS). Dairy small ruminants account for a minor part of the total agricultural output in France, Italy, and Spain (0.9 to 1.8%) and a larger part in Greece (8.8%). In FGIS, the dairy sheep industry is based on local breeds and crossbreeds raised under semi-intensive and intensive systems and is concentrated in a few regions in these countries. Average flock size varies from small to medium (140 to 333 ewes/farm), and milk yield from low to medium (85 to 216 L/ewe), showing substantial room for improvement. Most sheep milk is sold to industries and processed into traditional cheese types, many of which are Protected Denomination of Origin (PDO) cheeses for gourmet and export markets (e.g., Pecorino, Manchego, and Roquefort). By comparing break-even milk price among FGIS countries, we observed the following: (1) most Greek and French dairy sheep farms were unprofitable, with the exception of the intensive Chios farms of Greece; (2) milk price was aligned with cost of production in Italy; and (3) profitable farms coexisted with unprofitable farms in Spain. In FGIS, dairy goat production is based on local breeds raised under more extensive systems than sheep. Compared with sheep, average dairy goat herds are smaller (36 to 190 does/farm) but milk yield is greater (153 to 589 L/doe), showing room for improvement. Goat milk is mainly processed on-farm into dairy products for national markets, but some PDO goat milk cheeses (e.g., Murcia al Vino) are exported. Processed goat milk is sold for local human consumption or dehydrated for export. Mixed sheep-goat (e.g., Feta) and cow-sheep-goat milk cheeses are common in many countries. Strategies to improve the dairy sheep and goat sectors in these 4 countries are proposed and discussed.

Phase I trial of CYT997, a novel cytotoxic and vascular-disrupting agent.

BACKGROUND: CYT997 is a novel microtubule inhibitor and vascular-disrupting agent with marked preclinical anti-tumour activity. METHODS: This phase I dose-escalation study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of CYT997 administered by continuous intravenous infusion over 24 h every 3 weeks to patients with advanced solid tumours. RESULTS: Thirty-one patients received CYT997 over 12 dose levels (7-358 mg m(-2)). Doses up to 202 mg m(-2) were well tolerated. Dose-limiting toxicities were observed at 269 and 358 mg m(-2), consisting of grade 3 prolonged corrected QT interval in two patients and grade 3 hypoxia and grade 4 dyspnea in one patient. All toxicities were reversible. The pharmacokinetics of CYT997 were linear over the entire dose range. Dynamic contrast-enhanced magnetic resonance imaging scans showed significant changes in tumour K(trans) values consistent with vascular disruption in 7 out of 11 evaluable patients treated at CYT997 doses of >or=65 mg m(-2). Moreover, plasma levels of von Willebrand factor and caspase-cleaved cytokeratin-18 increased post-treatment at higher dose levels. Among 22 patients evaluable for response, 18 achieved stable disease for >2 cycles. CONCLUSIONS: CYT997 was well tolerated at doses that were associated with pharmacodynamic evidence of vascular disruption in tumours.

Incidence of severe nocturnal hypoglycemia in patients with type 1 diabetes treated with insulin lispro or regular human insulin in addition to basal insulin glargine.

BACKGROUND AND AIMS: Once-daily (OD) basal insulin glargine (GLA) can be used as part of a multiple daily injection regimen in patients with type 1 diabetes mellitus. This randomized, multicenter study compared GLA+prandial regular human insulin (RHI) with GLA+prandial insulin lispro (LIS) in reducing the incidence of severe nocturnal hypoglycemia at endpoint. In addition, the effects on glycemic control of both treatments were investigated. METHODS AND RESULTS: Patients (489) previously on neutral protamine Hagedorn (NPH) insulin or GLAR plus RHI/LIS were switched to, or continued on GLA (target fasting blood glucose [FBG]=5.0-6.7 mmol/L [90-120 mg/dL]) for 8 weeks (qualification phase) prior to randomization; patients continued with their previous bolus insulin. Patients (n=395) were then randomized to LIS (n=193) or RHI (n=202) and treated for 16 weeks. The proportion of patients experiencing severe nocturnal hypoglycemia at the end of the study was 1.55% (n=3) in the RHI group and 1.11% (n=2) in the LIS group (p=0.938 between groups); the mean difference was 0.44% (95% CI: -1.77, 2.21), suggesting non-inferiority of RHI versus LIS. At the end of the study, both treatments did not differ with respect to glycemic control, as measured by hemoglobin A(1c) and FBG. CONCLUSION: These results suggest that GLA+LIS and GLA+RHI treatments were associated with a similar and low rate of severe nocturnal hypoglycemia. Further studies with greater patient sizes are necessary to verify the findings from the current study.

Therapeutic mycophenolic acid monitoring by means of limited sampling strategy in orthotopic heart transplant patients.

Therapeutic drug monitoring (TDM) is essential to maintain the efficacy of many immunosuppressant drugs while minimizing their toxicity. TDM of mycophenolate mofetil requires area under the curve AUC determinations but appears laborious, costly, and clinically impractical. To overcome these problems, limited sampling strategies (LSS) have been proposed in adult and pediatric renal transplant patients. The purpose of this study was to develop an LSS in heart transplant patients. Forty-four mycophenolic acid (MPA) full AUC(0-12h) profiles were generated by high-performance liquid chromatography in nine heart transplant patients during the first 12 weeks posttransplant. Each patient received concomitant cyclosporine and prednisone therapy. Multiple stepwise regression analysis was used to define the time points of MPA levels to explain the MPA AUC(0-12h). Agreement between abbreviated AUC and the full AUC(0-12h) was tested by means of a Bland and Altman analysis. The highest coefficient of determination r(2) among MPA AUC and single concentrations (r(2) = .610) was observed with C(2), while C(12) provided the lowest one (r(2) = .003). Stepwise linear regression showed that the minimal model with the best estimation of MPA AUC(0-12h) was obtained at timed values of 1.25, 2, and 6 hours. The corresponding estimated model was AUC = 5.568 + 0.902 * C(1.25) + 2.022 * C(2) + 4.594 * C(6) (r(2) = .926). Bland and Altman analysis revealed good agreement between predicted AUC and full AUC. A further interesting model equation obtained by four samples was AUC = 3.800 + 1.015 * C(1.25) + 1.819 * C(2) + 1.566 * C(4) + 3.479 * C(6) (r(2) = .948).

Fusariosis associated with pathogenic fusarium species colonization of a hospital water system: a new paradigm for the epidemiology of opportunistic mold infections.

We sought the reservoir of Fusarium species in a hospital with cases of known fusarial infections. Cultures of samples from patients and the environment were performed and evaluated for relatedness by use of molecular methods. Fusarium species was recovered from 162 (57%) of 283 water system samples. Of 92 sink drains tested, 72 (88%) yielded Fusarium solani; 12 (16%) of 71 sink faucet aerators and 2 (8%) of 26 shower heads yielded Fusarium oxysporum. Fusarium solani was isolated from the hospital water tank. Aerosolization of Fusarium species was documented after running the showers. Molecular biotyping revealed multiple distinct genotypes among the isolates from the environment and patients. Eight of 20 patients with F. solani infections had isolates with a molecular match with either an environmental isolate (n=2) or another patient isolate (n=6). The time interval between the 2 matched patient-environment isolates pairs was 5 and 11 months, and 2, 4, and 5.5 years for the 3 patient-patient isolate pairs. The water distribution system of a hospital was identified as a reservoir of Fusarium species.

Simultaneous vulvar intraepithelial neoplasia and Paget's disease: report of two cases.

We describe a very rare association between intraepithelial, extramammary Paget's disease and human papillomavirus- (HPV) negative, keratinized type of VIN III observed in two elderly women. In both cases, morphological and immunohistochemical investigation showed two heterogeneous but intimately admixed neoplastic populations of vulvar epithelium. Atypical keratinocytes stained markedly and diffusely positive for high molecular weight cytokeratins, and moderately for p53 protein and c-erbB-2 immunostainings. Paget cells were diffusely positive for CEA, EMA, and low molecular weight cytokeratins, moderately and focally for c-erbB-2 and (in one case) for S-100. Morphological and immunohistochemical phenotypic differences between Paget cells and atypical keratinocytes suggest a simultaneous and incidental association of two distinct neoplastic disorders more than a mixed carcinoma in situ of vulvar epithelium.

Phenotypic heterogeneity influences apoptotic susceptibility to retinoic acid and cis-platinum of rat arterial smooth muscle cells in vitro: Implications for the evolution of experimental intimal thickening.

Rat aortic smooth muscle cells (SMCs) cultured from intimal thickening 15 days after endothelial injury (IT-15), unlike those of normal media, show a monolayered, epithelioid phenotype and high levels of cellular retinol binding protein-1 (CRBP). Epithelioid clones obtained from the normal media suggest a "mosaicism" of arterial SMCs. Intimal cell homeostasis from the balance of proliferation and apoptosis is critical for the progression of vascular lesions. All-trans retinoic acid (tRA) reduced [(3)H]thymidine incorporation and G(1)-->S phase progression of IT-15 and epithelioid clone but not of normal media and IT 60 days after injury (IT-60) SMCs. Hoechst staining, flow cytometry, and ligation-mediated polymerase chain reaction showed an increased susceptibility of IT-15 and epithelioid clone to tRA and cis-diaminedichloroplatinum II (CDDP)-induced apoptosis and cytotoxicity compared with normal media and IT-60 cells. The latter retained an increased susceptibility to tRA-induced apoptosis compared with normal media SMCs. tRA-induced apoptosis associated with an increased ratio of bax to bcl-2 by bax overexpression and cleavage of caspase-3. Anti-CRBP but not anti-IgG antibody prevented tRA-induced apoptosis and changes in related signaling molecules but not CDDP effects. Our findings support the relevant role of phenotypic heterogeneity in the determining proliferative as well as apoptotic behavior of arterial SMCs.

Dosage-dependent antifungal efficacy of V-echinocandin (LY303366) against experimental fluconazole-resistant oropharyngeal and esophageal candidiasis.

V-echinocandin (VER-002; LY303366) is a semisynthetic derivative of echinocandin B and a potent inhibitor of fungal (1, 3)-beta-D-glucan synthase. We studied the antifungal efficacy, the concentrations in saliva and tissue, and the safety of VER-002 at escalating dosages against experimental oropharyngeal and esophageal candidiasis caused by fluconazole-resistant Candida albicans in immunocompromised rabbits. Study groups consisted of untreated controls, animals treated with VER-002 at 1, 2.5, and 5 mg/kg of body weight/day intravenously (i.v.), animals treated with fluconazole at 2 mg/kg/day i.v., or animals treated with amphotericin B at 0.3 mg/kg/day. VER-002-treated animals showed a significant dosage-dependent clearance of C. albicans from the tongue, oropharynx, esophagus, stomach, and duodenum in comparison to that for untreated controls. VER-002 also was superior to amphotericin B and fluconazole in clearing the organism from all sites studied. These in vivo findings are consistent with the results of in vitro time-kill assays, which demonstrated that VER-002 has concentration-dependent fungicidal activity. Esophageal tissue VER-002 concentrations were dosage proportional and exceeded the MIC at all dosages. Echinocandin concentrations in saliva were greater than or equal to the MICs at all dosages. There was no elevation of serum hepatic transaminase, alkaline phosphatase, bilirubin, potassium, or creatinine levels in VER-002-treated rabbits. In summary, the echinocandin VER-002 was well tolerated, penetrated the esophagus and salivary glands, and demonstrated dosage-dependent antifungal activity against fluconazole-resistant esophageal candidiasis in immunocompromised rabbits.

Standardization of the comet assay technique on FRTL5 cells.

The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. The principle of break detection, using either the alkaline or neutral version of the assay, makes it a good technique for studying both double and single strand DNA breaks. Furthermore, the possibility of following DNA damage at different time moments also makes it possible to investigate the cell repair mechanisms. This explains why in the last few years there has been a tremendous increase in the number of laboratories which started to use this technique. The technique was first created for lymphocyte cells and later on has been used on many other cell types, growing both in suspension and adherent. To date, no one has applied this technique on normal differentiated endocrine cells, such as FRTL5 cells (Fisher Rat Thyroid Cells). The aim of this study has been to standardize the alkaline version of the Comet Assay technique on FRTL5 cells by studying the kinetics of DNA-damage and DNA-repair after different doses of UV-C (254 nm). FRTL-5 cells not only resulted very sensitive to UV-C (p<0.05 at 5 J/m2), but were also able to repair most of their DNA damage very rapidly (within one hour) as shown by a significant exponential regression in comet length. Finally, the successful measurement of biomarkers of UV-C on thyroid cells established the comet assay as a valuable tool in measurement of DNA damage and repair. Any radiation, or other damaging agents, interacting with living organisms could cause DNA damages which, depending upon dosages and kinetics of exposure, may or may not be completely repaired.

Radiation-dependent apoptosis on cultured thyroid cells.

Aim of this investigation is the study of the modifications and the DNA damage occurring in thyroid cells exposed to radiation. The FRTL-5 rat thyroid cell strain has been chosen for this study. Objects of this research are both Ionizing radiation, of fundamental interest for space missions, and the UV radiation, (also mutagen and frequent cause of several cancer forms). The present study of UV radiation represents a preliminarily tool to investigate the biological radiation damage. FRTL-5 cells have been irradiated with doses of UV-C (254 nm wavelength) ranging from 15 to 80 Joule/m2. The DNA damage has been analyzed with the 'DNA ladder by gel electrophoresis' technique. DNA has been extracted at 24 and 48 hours from irradiation. At 24h the apoptotic process is not detectable. At 48 h from irradiation, cells show the characteristic signs of apoptosis. The lower dose to which the apoptotic process is detectable, corresponds to 20 Joule/m2. At the higher doses a bigger percentage of cells undergoes apoptosis. These data confirms that the FRTL-5 biological system is particularly suitable for further studies on the biological mechanisms of radiation damage.

Opposing effects of protein kinase C and protein kinase A on metabotropic glutamate receptor signaling: selective desensitization of the inositol trisphosphate/Ca2+ pathway by phosphorylation of the receptor-G protein-coupling domain.

Signaling by the metabotropic glutamate receptor 1alpha (mGluR1alpha) can lead to the accumulation of inositol 1,4, 5-trisphosphate (InsP(3)) and cAMP and to the modulation of K(+) and Ca(2+) channel opening. At present, very little is known about how these different actions are integrated and eventually turned off. Unraveling the molecular mechanisms underlying these functions is crucial for understanding mGluR-mediated regulation of synaptic transmission. It has been shown that receptor-induced activation of the InsP(3) pathway is subject to feedback inhibition mediated by protein kinase C (PKC). In this study, we provide evidence for a differential regulation by PKC and protein kinase A of two distinct mGluR1alpha-dependent signaling pathways. PKC activation selectively inhibits agonist-dependent stimulation of the InsP(3) pathway but does not affect receptor signaling via cAMP. In contrast, protein kinase A potentiates agonist-independent signaling of the receptor via InsP(3). Furthermore, we demonstrate that the selectivity of PKC action on receptor signaling rests on phosphorylation of a threonine residue located in the G protein-interacting domain of the receptor. Modification at Thr(695) selectively disrupts mGluR1alpha-G(q/11) interaction without affecting signaling through G(s). Together, these data provide insight on the mechanisms by which selective down-regulation of a specific receptor-dependent signaling pathway can be achieved and on how cross-talk between different second messenger cascades may contribute to fine-tune short- and long-term receptor activity.

Transmission of an azole-resistant isogenic strain of Candida albicans among human immunodeficiency virus-infected family members with oropharyngeal candidiasis.

We report transmission of an azole-resistant, isogenic strain of Candida albicans in a human immunodeficiency virus (HIV)-infected family of two children with symptomatic oropharyngeal candidiasis and a mother with asymptomatic colonization over a 5-year period. These findings were confirmed by three different molecular epidemiology methods: interrepeat PCR, Southern hybridization with a C. albicans repetitive element 2 probe, and electrophoretic karyotyping. This study contributes to an evolving understanding of the mode of transmission of C. albicans, particularly in children, and underscores the importance of monitoring specimens from family members of HIV-infected patients.

Lack of Th2 cytokine increase during spontaneous remission of experimental allergic encephalomyelitis.

The mechanisms underlying spontaneous remission of autoimmune diseases are presently unknown, though regulatory T cells are believed to play a major role in this process. We tested the hypothesis that Th2 and/or other T cell regulatory cytokines cause the spontaneous remission of experimental allergic encephalomyelitis (EAE), a model of Th1-mediated autoimmunity. We analyzed the cytokine profile of lymph node and central nervous system-infiltrating cells in individual SJL mice at different stages of proteolipid protein (PLP) 139-151 peptide-induced EAE. We found that IFN-gamma slowly fades away after clinical recovery, whereas IL-4, IL-10 and transforming growth factor-beta remain low or undetectable. Our peptide-results therefore suggest that regulatory T cells producing anti-inflammatory cytokines are not involved in spontaneous remission of EAE and challenge the view that the Th1/Th2 balance has a key role in EAE regulation.

Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption.

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (10(8) and 10(7) CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10(8) CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P < or = 0.005), 10(7) CFU of C. neoformans (P < or = 0.05), and 10(7) CFU of A. fumigatus (P < or = 0.01). Yields were within the same range for 10(8) CFU of C. neoformans and l0(7) CFU of C. albicans for both HSCD extraction and PC extraction. For 10(7) CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P < or = 0.05). Yields obtained from 10(8) and 10(7) CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation.

On the basis of sequence homology and structural similarities, metabotropic glutamate receptors (mGluRs), extracellular Ca2+-sensing receptor, gamma-aminobutyric acid type B receptor, and pheromone receptors are enlisted in a distinct family within the larger G protein-coupled receptor superfamily. When expressed in heterologous systems, group I mGluRs can activate dual signal transduction pathways, phosphoinositides turnover and cAMP production. To investigate the structural basis of these coupling properties, we introduced single amino acid substitutions within the second and third intracellular loops (i2 and i3) of mGluR1alpha. Wild-type and mutant receptors were expressed in human embryonic kidney 293 cells and analyzed for their capacity to stimulate both signaling cascades. Each domain appeared to be critical for the coupling to phospholipase C and adenylyl cyclase. Within i2, Thr695, Lys697, and Ser702 were found to be selectively involved in the interaction with Gq class alpha subunit(s), whereas mutation of Pro698 and the deletion Cys694-Thr695 affected only Gs coupling. Furthermore, the mutation K690A profoundly altered mGluR1alpha signaling properties and imparted to the receptor the ability to couple to the inhibitory cAMP pathway. Within i3, we uncovered two residues, Arg775 and Phe781, that are crucial for coupling to both pathways, since their substitution leads to receptor inactivation.

New evidence that Candida albicans possesses additional ATP-binding cassette MDR-like genes: implications for antifungal azole resistance.

Emergence of resistance of Candida albicans to antifungal triazoles is increasingly recognized as an important cause of refractory mucosal candidiasis in HIV-infected patients. Recently, CDR1, which is thought to be analogous to the human MDR-1 P-glycoprotein, has been cloned in C. albicans. It has been proposed that its expression is partially responsible for fluconazole resistance in C. albicans. This gene is characterized by the presence of an ATP binding cassette (ABC) region and is distinct from the BENr gene which does not encode such a functional domain. As the molecular basis for fluconazole resistance appears to be multifactorial, we considered that there may be other ATP binding cassette-containing MDR genes that may potentially contribute to antifungal azole resistance in C. albicans. We therefore sought to identify potential target sequences that may be derived from candidate genes that share homology with the ATP binding cassette region of the human MDR-1 P-glycoprotein. Degenerate oligonucleotide primers based on the known sequence from the ATP binding cassette region of the human MDR-1 P-glycoprotein were used to amplify PCR products within the range of 100 bp in length from C. albicans isolates (3 fluconazole-susceptible and 3 fluconazole-resistant). Sequence analysis of individually subcloned PCR products, derived from the six isolates revealed 34 sequences in total. The results of our study identified 14 clones (with at least one per isolate) with a high degree of homology to the ATP binding cassette of the human MDR-1 P-glycoprotein. The BLAST search did not disclose homology of these new sequences to the C. albicans CDR1 gene, suggesting that C. albicans may possess more than one MDR-like gene. We conclude that C. albicans may possess one or more additional genes encoding ATP binding cassette MDR-like proteins that are distinct from CDR 1 and which could participate in the development of fluconazole resistance.

PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi.

The application of PCR technology to molecular diagnostics holds great promise for the early identification of medically important pathogens. PCR has been shown to be useful for the detection of the presence of fungal DNA in both laboratory and clinical samples. Considerable interest has been focused on the utility of selecting universal primers, those that recognize constant regions among most, if not all, medically important fungi. Once an amplicon, or piece of amplified DNA determined by the unique pair of oligonucleotide primers, has been generated, several different methods may be used to distinguish between genera and between species. The two major approaches have utilized differences in restriction enzyme digestion patterns or hybridization with specific probe. We report the application of single-strand conformational polymorphism (SSCP) as a technique to delineate the differences between fungal species and/or genera. Minor sequence variations in small single-stranded DNA cause subtle changes in conformation, allowing these strands to be separated on polyacrylamide gels by SSCP. We used a 197-bp fragment amplified from the 18S rRNA gene, common to all medically important fungi. After amplification, the fragments were denatured and run on an acrylamide-glycerol gel at room temperature or 4 degrees C for 4.5 or 4 h, respectively. Under room temperature conditions, the SSCP patterns for Candida albicans, Candida tropicalis, and Candida parapsilosis were identical and all strains within each species demonstrated the same pattern. These patterns differed markedly from those of the genus Aspergillus. The SSCP patterns of major and minor bands at room temperature permitted distinction between strains of Aspergillus fumigatus and Aspergillus flavus. There also was consistency of the SSCP banding pattern among different strains of the same Aspergillus species. The SSCP patterns for other medically important opportunistic fungi, such as Cryptococcus neoformans, Pseudallescheria boydii, and Rhizopus arrhizus, were sufficiently unique to permit distinction from those of C. albicans and A. fumigatus. We conclude that the technique of PCR-SSCP provides a novel method by which to recognize and distinguish medically important opportunistic fungi and which has potential applications to molecular diagnosis, taxonomic classification, molecular epidemiology, and elucidation of mechanisms of antifungal drug resistance.

Therapeutic monitoring of experimental invasive pulmonary aspergillosis by ultrafast computerized tomography, a novel, noninvasive method for measuring responses to antifungal therapy.

Pulmonary infiltrates in neutropenic hosts with invasive aspergillosis are due to vascular invasion and hemorrhagic infarction. In order to measure the effect of antifungal compounds on this organism-mediated tissue injury, we monitored the course of pulmonary infiltrates by serial ultrafast computerized tomography (UFCT) in persistently granulocytopenic rabbits with experimental invasive pulmonary aspergillosis. The course of pulmonary lesions measured by serial UFCT scans was compared with those measured by conventional chest radiography, histopathological resolution of lesions, and microbiological clearance of Aspergillus fumigatus. Treatment groups included either amphotericin B colloidal dispersion in dosages of 1, 5, and 10 mg/kg of body weight per day intravenously or conventional desoxycholate amphotericin B at 1 mg/kg/day intravenously. Therapeutic monitoring of pulmonary lesions by UFCT demonstrated a significant dose-response relationship. Lesions continued to progress in untreated controls, whereas lesions in treated rabbits initially increased and then decreased in response to antifungal therapy in a dosage-dependent manner (P < or = 0.05 to P < or = 0.005, depending upon the groups compared). This same trend of resolution of lesions in response to antifungal therapy was also demonstrated by postmortem examination and by microbiological clearance of the organism. These data indicated that amphotericin B colloidal dispersion at 5 and 10 mg/kg/day exerted a more rapid rate of clearance of lesions than conventional amphotericin B. UFCT was more sensitive than conventional chest radiography in detecting lesions due to invasive pulmonary aspergillosis (P < 0.05 to P < 0.005, depending upon the groups compared). These findings establish a correlation among UFCT-defined lesions, microbiological response, and resolution of pathologically defined lesions in experimental invasive pulmonary aspergillosis. Serial monitoring of UFCT-defined lesions of aspergillosis provides a novel system for determining the antifungal response of organism-mediated tissue injury.