Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

Photosynthetic activity of intertidal microphytobenthic communities during emersion: in situ measurements of chlorophyll fluorescence (PAM) and CO2 flux (IRGA)1

Photosynthetic microphytobenthic activity has increasingly been examined using pulse‐amplitude‐modulated (PAM) fluorescence techniques. Nevertheless, estimating carbon production rates from fluorescence measurements implies the establishment of reliable relationships. The aim of this study was to determine such a relationship from field measurements of both PAM fluorescence and CO₂ fluxes. Three study sites of varying sedimentary features were investigated in different seasons. Both linear and with plateau relationships were obtained between the fluorescence parameter (relative electron transport rate [rETR]) and the community‐level carbon‐fixation rate (gross community primary production rate [GCP] in mg C · m^?2 · h^?1). The correlation calculated from the whole data set (i.e., all sites and all seasons) was very strong (n = 106; r = 0.928). Significant correlations were also obtained for light‐curve parameters assessed with the two methods: P_m (n = 8; r = 0.920) and I_k (n = 8; r = 0.818). Total community‐level carbon fixation for the emersion period was calculated from fluorescence measurements according to the relationship established between GCP and rETR, and between light‐curve parameters, and the results were compared to the estimation obtained directly from GCP measurements. The agreement between the two estimations was quite good for both ways of calculation (with a mean discrepancy of 30% for the first one and ?2% for the second one). These results suggest the potential application of PAM measurements to calculate carbon‐fixation rates at large spatial and temporal scales, provided that a set of experiments coupled with CO₂‐flux measurements are performed.     

Determination of fluoxetine in pharmaceutical and biological samples based on the silver nanoparticle enhanced fluorescence of fluoxetine–terbium complex

In this study, a simple and sensitive spectrofluorimetric method is presented for the determination of fluoxetine based on the enhancing effect of silver nanoparticles (AgNPs) on the terbium–fluoxetine fluorescence emission. The AgNPs were prepared by a simple reduction method and characterized by UV–Vis spectroscopy and transmission electron microscopy. It was indicated that these AgNPs have a remarkable amplifying effect on the terbium‐sensitized fluorescence of fluoxetine. The effects of various parameters such as AgNP and Tb³⁺ concentration and the pH of the media were investigated. Under obtained optimal conditions, the fluorescence intensity of the terbium–fluoxetine–AgNP system was enhanced linearly by increasing the concentration of fluoxetine in the range of 0.008 to 19 mg/L. The limit of detection (b + 3s) was 8.3 × 10^‐4 mg/L. The interference effects of common species found in real samples were also studied. The method had good linearity, recovery, reproducibility and sensitivity, and was satisfactorily applied for the determination of fluoxetine in tablet formulations, human urine and plasma samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of new apolipoprotein B epitopes and haplotypes and their distribution in swine populations

Results from comparative immunogenetic studies on inheritance and identification of four new apolipoprotein B (apoB) allotypes and three additional apoB haplotypes and their distribution in miniature and domestic swine are presented. Immunological surveys on the four new and 16 previously described Lpb allotypes and genetic analysis of their segregation in progenies, of miniature and domestic swine and their crosses, indicate that three new allotypes designated Lpb9, Lpb10 and Lpb101 are individual (mutant) apoB epitopes, each representing a discriminating marker for one of the new apoB haplotypes specified by three new apoB alleles designated Lpb⁹, Lpb¹⁰ and Lpb¹⁰¹. The fourth allotype, Lpb20, is one of the common epitopes forming the alternative epitope pair with Lpb10, and is a constituent of each of the eight previously described and two new apoB haplotypes. The new apoB alleles have so far been found only in miniature swine, with Lpb¹⁰ being the most frequent in the Göttingen, Vietnamese Potbelly and Japanese Miniature, Lpb⁹ was detected only in Minnesota Miniature and Lpb101 only in Vietnamese Potbelly. The common allotype, Lpb20, shares immunological similarities with human apoB indicating its ancestral origin, whereas none of the alloreagents detecting the three individual apoB variants, Lpb9, Lpb10 or Lpb101, showed cross-reactivity with human apoB, suggesting their exclusive swine origin and evolvement during speciation through mutations.     

Highly sensitive spectrofluorimetric determination of trace amounts of prulifloxacin using the aluminium(III)–prulifloxacin system

The fluorescence of the prulifloxacin (PUFX)–Al(III) system was investigated . Experiments indicated that the fluorescence intensity of prulifloxacin could be greatly enhanced by Al(III) and sensitized by sodium dodecylbenzene sulphonate (SDBS). Accordingly, a sensitive spectrofluorimetric method for the determination of prulifloxacin was established. While excited at 275 nm, the enhanced fluorescence intensity at 412 nm of the system (ΔF) showed a good linear relationship with the concentration of prulifloxacin within the range 4.0 × 10^–8–3.0 × 10^–6 mol/L. The regression equation was ΔF = 9.83 + 10.8 × 10⁷c (mol/L); the correlation coefficient and detection limit (3σ/k) were 0.99901 and 2.0 × 10^–8 mol/L, respectively. The proposed method has been successfully applied to determine prulifloxacin in real pharmaceutical samples. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma     

Microbial diagnostic microarray for food‐ and water‐borne pathogens

A microbial diagnostic microarray for the detection of the most relevant bacterial food- and water-borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 10⁴ cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray-based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data.     

The infectivity and host range of Orgyia anartoides nucleopolyhedrovirus

The painted apple moth (PAM), Teia anartoides (Walker) (Lepidoptera: Lymantriidae) made a recent incursion into New Zealand. A nucleopolyhedrovirus (NPV), Orgyia anartoides NPV (OranNPV), originally isolated from PAM in Australia, was tested for its pathogenicity to PAM and a range of non‐target insect species found in New Zealand, to evaluate its suitability as a microbial control for this insect invader. Dosage‐mortality tests showed that OranNPV was highly pathogenic to PAM larvae; mean LT₅₀ values for third instars ranged from 17.9 to 8.1 days for doses from 10² to 10⁵ polyhedral inclusion bodies/larva, respectively. The cause of death in infected insects was confirmed as OranNPV. Molecular analysis established that OranNPV can be identified by PCR and restriction digestion, and this process complemented microscopic examination of infected larvae. No lymantriid species occur in New Zealand; however, the virus had no significant effects on species from five other lepidopteran families (Noctuidae, Tortricidae, Geometridae, Nymphalidae and Plutellidae) or on adult honeybees. Thus, all indications from this initial investigation are that OranNPV would be an important tool in the control of PAM in a future incursion of this species into New Zealand.     

Polymer composite fluorescent hydrogel film based on nitrogen‐doped carbon dots and their application in the detection of Hg2+ ions

A simple microwave‐assisted solvothermal method was used to prepare fluorescent nitrogen‐doped carbon dots (N‐CDs) with high fluorescence quantum yield (79.63%) using citric acid and N‐(2‐hydroxyethyl)ethylenediamine as starting materials. The PVAm‐g‐N‐CDs grafted products were synthesized by amide bond formation between the carboxylic groups of N‐CDs and amine groups of polyvinylamine (PVAm). Fluorescent hydrogel films (PVAm‐g‐N‐CDs/PAM) were synthesized by interpenetration polymer network polymerization of PVAm‐g‐N‐CDs and acrylamide (AM). When used for ion detection, we found that the fluorescence of the hydrogel films was clearly quenched by addition of Hg²⁺. Repeatability tests on using the hydrogel films for Hg²⁺ detection showed that they could be applied at least three times. The PVAm‐g‐N‐CDs/PAM could serve as an effective fluorescent sensing platform for sensitive detection of Hg²⁺ ions with a detection limit of 0.089 μmol/L. This work may offer a new approach for developing recoverable and sensitive N‐CDs‐based sensors for biological and environmental applications.     

Spectrofluorimetric determination of bilirubin in serum samples

A new spectrofluorimetric method was developed for determination of trace amount of bilirubin. Using oxytetracycline–Eu³⁺ as a fluorescent probe, in the buffer solution of pH = 7.3, bilirubin can reduce remarkably the fluorescence intensity of the oxytetracycline–Eu³⁺ complex at λ = 612 nm and the reduced fluorescence intensity was in proportion to the concentration of bilirubin. Optimum conditions for the determination of bilirubin were also investigated. The linear range and limit of detection for the determination of bilirubin were 5.0 × 10^?7, 3.0 × 10^?5 and 7.7 × 10^?8 mol L^?1, respectively. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess bilirubin in serum samples and compared with the modified Jendrassik–Grof method in clinical analysis. The quenching mechanism of the fluorescence intensity in the system is also discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

TEMPERATURE EFFECTS ON MICROALGAL PHOTOSYNTHESIS‐LIGHT RESPONSES MEASURED BY O2 PRODUCTION,PULSE‐AMPLITUDE‐MODULATED FLUORESCENCE,AND 14C ASSIMILATION1

Short‐term temperature effects on photosynthesis were investigated by measuring O₂ production, PSII‐fluorescence kinetics, and ¹⁴C‐incorporation rates in monocultures of the marine phytoplankton species Prorocentrum minimum (Pavill.) J. Schiller (Dinophyceae), Prymnesium parvum f. patelliferum (J. C. Green, D. J. Hibberd et Pienaar) A. Larsen (Coccolithophyceae), and Phaeodactylum tricornutum Bohlin (Bacillariophyceae), grown at 15°C and 80 μmol photons · m^?2 · s^?1. Photosynthesis versus irradiance curves were measured at seven temperatures (0°C–30°C) by all three approaches. The maximum photosynthetic rate (P^C_max) was strongly stimulated by temperature, reached an optimum for Pro. minimum only (20°C–25°C), and showed a similar relative temperature response for the three applied methods, with Q₁₀ ranging from 1.7 to 3.5. The maximum light utilization coefficient (α^C) was insensitive or decreased slightly with increasing temperature. Absolute rates of O₂ production were calculated from pulse‐amplitude‐modulated (PAM) fluorometry measurements in combination with biooptical determination of absorbed quanta in PSII. The relationship between PAM‐based O₂ production and measured O₂ production and ¹⁴C assimilation showed a species‐specific correlation, with 1.2–3.3 times higher absolute values of P^C_max and α^C when calculated from PAM data for Pry. parvum and Ph. tricornutum but equivalent for Pro. minimum. The offset seemed to be temperature insensitive and could be explained by a lower quantum yield for O₂ production than the theoretical maximum (due to Mehler‐type reactions). Conclusively, the PAM technique can be used to study temperature responses of photosynthesis in microalgae when paying attention to the absorption properties in PSII.     

Interaction of glucose‐derived carbon quantum dots with silver and gold nanoparticles and its application for the fluorescence detection of 6‐thioguanine

The interaction of glucose‐derived carbon quantum dots (CQDs) with silver (Ag) and gold (Au) nanoparticles (NPs) was explored by fluorescence spectroscopy. Both metal NPs cause an efficient quenching of CQD fluorescence, which is likely due to the energy transfer process between CQDs as donors and metal NPs as acceptors. The Stern–Volmer plots were evaluated and corresponding quenching constants were found to be 1.9 × 10¹⁰ and 2.2 × 10⁸ M^?1 for AgNPs and AuNPs, respectively. The analytical applicability of these systems was demonstrated for turn‐on fluorescence detection of the anti‐cancer drug, 6‐thioguanine. Because the CQD–AgNP system had much higher sensitivity than the CQD–AuNP system, we used it as a selective fluorescence probe in a turn‐on assay of 6‐thioguanine. Under optimum conditions, the calibration graph was linear from 0.03 to 1.0 μM with a detection limit of 0.01 μM. The developed method was applied to the analysis of human plasma samples with satisfactory results.     

A fluorescent compound in oat root plasma membrane

Homogenates of 7-day-old oat (Avena sativa L. cv. Brighton) roots were highly fluorescent (excitation and emission maxima around 360 and 440 nm, respectively). Less than ¹/₁₀ as much fluorescence per g fresh weight was found in oat shoots or in wheat (Triticum aestivum L. cv. Drabant) roots or shoots. Most of the fluorescence of oat roots was found in the soluble fraction (150 000g supernatant). However, some could be detected in the plasma membrane fraction (excitation and emission maxima 365 and 417 nm, respectively), which contained a 3-fold higher fluorescence per mg protein than the homogenate. Growth of oat or wheat in a medium containing, 10-^?5M scopoletin (6-methoxy-7-hy-droxy coumarin), a fluorescent compound previously reported to be present in both wheat and oat roots, caused the disappearance of scopoletin from the medium (proportional to the amount of roots) and the appearance of increased fluorescence in the root homogenates but not in the shoot homogenates. In both oat and wheat roots ail of the extra fluorescence was recovered in the soluble fraction and at least in wheat it consisted of unconverted scopoletin. The concentration of scopoletin in wheat roots grown in 10-^?5M scopoletin was around 50 nmol (g fresh weight)^?1, or about five times the concentration in the growth medium. Scopoletin in the growth medium (10-^?5M) or in the assays (up to 10-^?4M) did not affect Mg²⁺-, Mg²⁺+K⁺- or Ca²⁺-ATPase activities in wheat or oat roots. The fluorescence properties of the oat plasma membrane were different from those of authentic scopoletin. Either the surroundings modify the fluorescence of membrane-associated scopoletin or the endogenous fluorescent compound is not scopoletin but a glycoside-derivative of scopoletin or some completely unrelated compound.     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 μg diuron l⁻¹. Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 μg l⁻¹ defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 μg chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 μmol quanta m⁻² s⁻¹ with Phaeodactylum and thylakoids, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spectroscopic characterization,calorimetric study and molecular docking to evaluate the bioconjugation of maltol with hemoglobin

Maltol, a food additive, is extensively used in our daily life. To date, its biological safety is still debated. In this article, binding interaction of maltol with bovine hemoglobin (BHb), an important functional protein, was studied by molecular docking research and spectroscopic and calorimetric measurements. We found that maltol could cause structural changes of BHb. By interacting with Glu 101 (1.27 Å) and Lys 104 (2.49 Å) residues, maltol changed the cavity structure and induced a microenvironment change around tryptophan (Trp) residue. Thermodynamic parameters obtained from isothermal titration calorimetry (ITC) measurement showed that hydrophobic forces were the main forces existing in this system. The association constant of K (8.0 ± 3.4 × 10⁴ M^?1) shows the mild ligand–protein binding for maltol with BHb. The α‐helix amount in BHb increased (59.6–62.6%) with different concentrations of maltol and the intrinsic fluorescence intensity was quenched by maltol, indicating the conformation changes and denaturation of BHb. This work presents the interactions of maltol with BHb at the molecular level and obtains evidence that maltol induces adverse effects to proteins in vitro.     

Experimental pulmonary candidiasis

The initial interaction of Candida albicans with pulmonary tissue of B6D2/F₁ mice was investigated. The LD₅₀ for mice challenged intravenously (IV) was approximately 3 × 10⁵ yeasts, whereas the LD₅₀ by the intratracheal (IT) route was in excess of 10⁸ yeasts. Mice challenged IV died of progressive yeast growth in the kidneys. In contrast, mice challenged IT rapidly eliminated the entire inoculum by the first day after challenge. Resident pulmonary alveolar macrophages (PAM) killed upwards of 70% of C. albicans in an in vitro killing assay. At effector: target ratios favoring the effector cell population resident PAM were able to restrict the formation of yeast germ tubes to only 30% of the yeasts, whereas at equivalent ratios virtually all of the intracellular yeasts produced germ tubes. Evaluation of the ability of PAM, harvested from genetically different strains of inbred mice, to kill C. albicans in vitro showed that killing ability was a property of resident PAM from mice with the black 6 background. It was discovered that during the initial stages of infection in vivo, the expression of the F4/80 surface molecule was down regulated, and the expression of the Mac 1 surface molecule upregulated. There were no quantitative changes in expression of either Mac 2, Mac 3, Ly 5 or the 5C6 surface epitopes. Taken together, the data show that pulmonary tissue is quantitatively very resistant to C. albicans infection, because of the ability of resident PAM to rapidly phagocytize and kill yeasts. Killing of C. albicans by resident PAM may be a property of a subset of this mononuclear phagocyte population and was accompanied by alterations in the expression of surface molecules.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

The relative contribution of resident pulmonary alveolar macrophage and inflammatory polymorphonuclear neutrophils in host resistance to pulmonary infection by Candida albicans

Cortisone (CA) or cyclophosphamide (Cy) treatment of mice was used to investigate the relative contributions of pulmonary alveolar macrophages (PAM) and inflammatory neutrophils (PMN) in the initial defense against intratracheal challenge (IT) with Candida albicans. Mice treated with either CA or Cy were susceptible to IT challenge with 10–100 x less C. albicans than were untreated mice. Untreated mice rapidly eliminated C. albicans from their lungs with the majority of the organisms being cleared within three hours of challenge. Mice treated with CA initially cleared some of the C. albicans but were unable to clear all the C. albicans as did the untreated mice. Mice treated with Cy were unable to clear C. albicans from their lungs. Candida albicans did not disseminate from the lungs of untreated mice, while in both of the treated groups, C. albicans disseminated to the liver, spleen, brain and kidneys, rapidly killing the treated hosts. Analysis of the changes in cells in lung lavage fluids collected at various times after C. albicans challenge, revealed that large numbers of PMN accumulated in the lungs of both untreated and CA-treated mice, whereas PMN were virtually undetectable in lavage fluids from Cy-treated mice. Resident PAM from untreated mice were able to kill approximately 70 % of 10⁵ C. albicans in a 3 hr in vitro killing assay. By contrast, at similar effector: target ratios, resident PAM from Cy-treated mice killed only about 20% of the inoculum and resident PAM from CA-treated mice were unable to kill C. albicans. PMNs from both untreated and CA-treated mice killed approximately 70% of 10⁵ C. albicans in vitro. The data indicates that both PAM and PMN were critical to the initial clearance of C. albicans from pulmonary tissue. The accumulation of PMN in the lungs appeared to be required for the complete clearance of C. albicans from the lungs yet was not sufficient to inhibit dissemination of C. albicans from the lungs in CA-treated mice. The presence of PAM with in vitro candidacidal abilities appeared to be required for both the clearance of C. albicans and inhibition of dissemination of C. albicans from the lungs. Compromise of either PAM or PMN function can lead to increased pulmonary susceptibility to C. albicans.