Calcofluor White Staining Research Articles

Functional Sieve Element Protoplasts

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.

The Value of Computed Tomography-Guided Percutaneous Lung Biopsy for Diagnosis of Invasive Fungal Infection in Immunocompromised Patients

We assessed Calcofluor white staining, Aspergillus polymerase chain reaction, and a galactomannan enzyme immunoassay for diagnosis of fungal infection with use of computed tomography-guided percutaneous lung biopsy specimens obtained from 61 patients. The sensitivity and specificity of computerized tomography, Aspergillus polymerase chain reaction, and galactomannan enzyme immunoassay were 100% and 50%, 100% and 86%, and 88% and 94%, respectively.

Identification of Chitin in the Prismatic Layer of the Shell and a Chitin Synthase Gene from the Japanese Pearl Oyster,Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.

Flumorph Is a Novel Fungicide That Disrupts Microfilament Organization in Phytophthora melonis

ABSTRACT The mechanism of the effects of flumorph (a novel fungicide) was investigated by analyzing alterations of hyphal morphology, cell wall deposition patterns, F-actin organization, and other organelles in Phytophthora melonis. Calcofluor white staining suggested that flumorph did not inhibit the synthesis of cell wall materials, but disturbed the polar deposition of newly synthesized cell wall materials during cystospore germination and hyphal growth. After exposure to flumorph, zoospores were able to switch into cystospores accompanied with the formation of a cell wall, whereas cystospores failed to induce the isotropic-polar switch and did not produce germ tubes but continued the isotropic growth phase. In flumorph-treated hyphae, the most characteristic change was the development of periodic swelling ("beaded" morphology) and the disruption of tip growth. Newly synthesized cell wall materials were deposited uniformly throughout the diffuse expanded region of hyphae, in contrast to their normal polarized patterns of deposition. These alterations were the result of F-actin disruption, identified with the fluorescein isothiocynate (FITC)-phalloidin staining. The disruption of F-actin also was accompanied by disorganized organelles: each swelling of subapical hyphae was associated with a nucleus. Vesicles did not undergo polarized secretion to the apical hyphae, but diffused around nuclei for the subapical growth; thus, the cell wall was thickened with periodic expansion along the hyphae. Upon removing flumorph, normal tip growth and organized F-actin were observed again. These data, as well as data published earlier, suggest that flumorph may be involved in the impairment of cell polar growth through directly or indirectly disrupting the organization of F-actin. The primary site of action by flumorph in the disruption of the F-actin organization is under investigation.

First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera)

The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals.

Eosinophilic Pneumonia in a Traveller Returning from Mexico

A previously healthy 45-year-old Korean-born man presented with pleuritic chest pain and a dry cough in February 2005. His symptoms began one week after returning home to Toronto, Ontario, following a trip to northern Mexico, where he had spent several days in a large city attending meetings. He denied having fever, rash or arthralgia. His chest pain and cough were initially managed symptomatically with an analgesic and a cough supressant. However, the symptoms persisted and after one week, he was referred to a respirologist for further investigation and management. His physical examination was normal. His complete blood count revealed marked eosinophilia (6.0×109/L) and his chest radiograph documented a right lung infiltrate. A computed tomography scan confirmed consolidation in the right upper lobe and revealed centrilobular nodules in a tree-in-bud pattern, right hilar and mediastinal lymphadenopathy and small nodules (less than 0.5 mm) scattered throughout both lungs. He underwent bronchoscopic alveolar lavage and transbronchial biopsy. Cytology of the bronchoscopic alveolar lavage fluid revealed marked eosinophilia. Gram stain and calcofluor white (fungal) stain were negative. Histopathology of the biopsy documented nonspecific eosinophilic bronchitis. There were no identifiable organisms with hematoxylin and eosin, periodic acid-Schiff or GrocottGomori methenamine-silver stains of the tissue. A microbiological test was performed. What was the diagnosis and how was it made?

Species distribution and antifungal susceptibilities of dermatophytes during a one year period at a university hospital in Turkey

Dermatophyte infections have been considered to be a major public health problem in many parts of the world. The aim of this study was to determine the causative agents of dermatophytoses and their antifungal susceptibilities in a Turkish University Hospital, west of Turkey. A total of 926 patients suspected to have dermatophytic lesions were examined over a period of 1 year (2001-2002). Samples collected from skin, hair and nails were submitted to direct microscopical examination using KOH and Calcofluor white stain, cultured on Sabouraud dextrose agar and Mycosel agar. The prevalence of dermatophytoses was 7.34% (68/926). Trichophyton rubrum was the most frequent dermatophyte isolated (56%) followed by T. mentagrophytes (38%), T. violaceum (1.5%), T. verrucosum (1.5%), Microsporum canis (1.5%) and Epidermophyton floccosum (1.5%). Tinea pedis (47%) was the most common type of infection, followed by tinea unguium (29%), tinea inguinalis (15%), tinea corporis (7.4%) and tinea capitis (1.6%). Secondary, we have tested 68 strains of dermatophytes against four antifungal agents following mainly the National Committee for Clinical Laboratory Standards M38-P standard for filamentous fungi. In general, all antifungals were shown to be highly effective and itraconazole and naftifine appeared more active than ketoconazole and oxiconazole.

Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.

A real-time polymerase chain reaction assay for detection of Pneumocystis from bronchoalveolar lavage fluid

Pneumocystis jiroveci is an important cause of pneumonia in immunocompromised individuals. This organism cannot be cultured, and therefore, diagnosis relies on microscopic identification of the organism using stains or antibodies. Although simple, these tests are insensitive and require expertise for accurate interpretation. We developed a real-time polymerase chain reaction (PCR) assay that provides sensitive and objective detection of Pneumocystis from bronchoalveolar lavage fluid. Primers and fluorescence resonance energy transfer probes were developed that target the cdc2 gene of P. jiroveci. Assay sensitivity is 6 copies of target per microliter of sample. No cross-reactivity occurs with other pathogens, and the PCR assay has a 21% increase in clinical sensitivity as compared with Calcofluor white staining. The real-time PCR assay provides a sensitive, rapid, and objective method for the detection of Pneumocystis from bronchoalveolar lavage fluid.

Assessment of a Novel Approach to Evaluate the Outcome of Endoscope Reprocessing

To investigate and evaluate the use of a portable luminometer system for detecting contamination following the reprocessing and high-level disinfection of flexible endoscopes.Random sampling of endoscopes spaced at 1- to 2-week intervals following normal use in patients.Portable luminometer system testing of 31 endoscopes undergoing reprocessing, 63 stored endoscopes, and 15 reprocessed endoscopes that underwent in-depth microbiological analysis. For testing with the portable luminometer system, samples were collected by swabbing a 100-cm2 shank surface area and the internal tip end orifice. Standardization of portable luminometer system results was performed in vitro by comparison of serial dilutions of known quantities of microorganisms and blood, tested before and after sterilization by autoclave. Microbiological analysis included Gram stain, culture for aerobic bacteria, and gene probes for Mycobacterium tuberculosis, herpes simplex viruses 1 and 2, and Cytomegalovirus. Trichrome and calcofluor white stains were used to detect parasites and fungi. Legionella was detected by stain with fluorescent-labeled monoclonal antibody.The gastroendoscopy unit of a Veterans Affairs hospital.The portable luminometer system was capable of detecting microbial and cellular contamination of flexible endoscopes following high-level disinfection and reprocessing. The sensitivity of the assay was sufficient for detecting low-level contamination.The system provided a rapid microbiological outcome monitor for the cleaning and disinfection process. The system was easy to use and relatively accurate.

Role of chitin synthase genes in Fusarium oxysporum.

Three structural chitin synthase genes, chs1, chs2 and chs3, were identified in the genome of Fusarium oxysporum f. sp. lycopersici, a soilborne pathogen causing vascular wilt disease in tomato plants. Based on amino acid identities with related fungal species, chs1, chs2 and chs3 encode structural chitin synthases (CSs) of class I, class II and class III, respectively. A gene (chs7) encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from Saccharomyces cerevisiae, an endoplasmic reticulum (ER) protein required for the export of ScChs3p (class IV) from the ER. So far no CS gene belonging to class IV has been isolated from F. oxysporum, although it probably contains more than one gene of this class, based on the genome data of the closely related species Fusarium graminearum. F. oxysporum chs1-, chs2- and chs7-deficient mutants were constructed through targeted gene disruption by homologous recombination. No compensatory mechanism seems to exist between the CS genes studied, since chitin content determination and expression analysis of the chs genes showed no differences between the disruption mutants and the wild-type strain. By fluorescence microscopy using Calcofluor white and DAPI staining, the wild-type strain and Deltachs2 and Deltachs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the Deltachs1 mutant showed compartments containing up to four nuclei. Pathogenicity assays on tomato plants indicated reduced virulence of Deltachs2 and Deltachs7 null mutants. Stress conditions affected normal development in Deltachs2 but not in Deltachs1 or Deltachs7 disruptants, and the three chs-deficient mutants showed increased hyphal hydrophobicity compared to the wild-type strain when grown in sorbitol-containing medium. The chitin synthase mutants will be useful for elucidating cell wall biogenesis in F. oxysporum and the relationship between fungal cell wall integrity and pathogenicity.

Adaptation of FUN-1 and Calcofluor white stains to assess the ability of viable and nonviable yeast to adhere to and be internalized by cultured mammalian cells

The FUN-1 and Calcofluor white stains can be used in concert to assess the ability of viable and nonviable yeast to adhere to, and be internalized by, host mammalian cells in vitro. With this method, only extracellular yeast stain with Calcofluor, dead yeast cells have diffuse cytoplasmic yellow–green fluorescence, and live yeast have cytoplasmic orange–red or yellow–orange fluorescent intravacuolar structures.

Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods.

We examined four staining methods on replicate smears of 313 respiratory specimens submitted for Pneumocystis jiroveci examination. The sensitivity and specificity of Calcofluor white stain (CW) were 73.8 and 99.6%, respectively. The sensitivity and specificity of Grocott-Gomori methenamine silver stain (GMS) were 79.4 and 99.2%, respectively. The sensitivity and specificity of Diff-Quik stain were 49.2 and 99.6%, respectively. The sensitivity and specificity of Merifluor Pneumocystis stain were 90.8 and 81.9%, respectively. Only CW and GMS had positive and negative predictive values of >90%.

056 In Vitro Evaluation of Biofilm Development on Tissue Engineered Skin

Bacteria reside in various forms, including planktonic, or free‐floating, or as biofilms, which are a tightly adherent, potentially more treatment‐resistant form. Biofilms, composed in part as polysaccharide matrices, can act as protection for bacteria by shielding them from antimicrobials and the innate immune system. It is estimated that bacterial biofilm are responsible for 65–80% of all chronic infections and may be responsible in part for non‐healing of chronic wounds.An advance in the treatment of non‐healing wounds has lead to the development of human skin equivalents (HSE), which can successfully treat many non‐healing wounds. Better understanding the interaction between bacteria and HSE might improve patient outcomes. Studies involving planktonic E. coli and HSE have demonstrated the growth of bacterial colonies only on the dermal surface. Growth was not seen on the epidermal layer of the HSE, possibly due in part to the expression of innate antimicrobial peptides called human β defensin‐2 (hBD‐2), which are expressed by HSE keratinocytes. The objective of this study was to determine if biofilm formation could occur on a bioengineered HSE.We used a commercially available HSE. Three (3) mm full‐thickness incisions were made in a triangular section of HSE. Each section was inoculated on the epidermal aspect with 1.0 × 105CFU per gram of Pseudomonas aeruginosa at 35 degrees Celsius for specified time points. Sections of HSE were sampled in areas of injury at various time points. Biopsy sections were processed for histologic analysis with H&E and epifluorescent microscopy to visualize Pseudomonas aeruginosa. We found that biofilm formation occurred at multiple time points on HSE. Colonies of adherent bacteria were visualized within the injured area of the epidermal layer of HSE by H & E staining. Eplifluoresecnt microscopy using calcofluor white staining revealed the characteristic exopolysaccharide (EPS) matrix of biofilm.Visualization of the expression of human β defensin‐2 (hBD‐2) with HSE after biofilm formation is underway. This knowledge will help to understand the role of bacterial biofilms within HSE and their effect on subsequent healing.

Prevalence of Microsporidian Spores Shed by Asymptomatic Lovebirds: Evidence for a Potential Emerging Zoonosis

Several reports implicate Encephalitozoon hellem, a microsporidian parasite first described in humans and later in birds, as the causative agent of severe disease in immunocompromised patients. This study was conducted to assess the prevalence of microsporidian spores shed in the droppings of lovebirds. During a 7-month period, a total of 198 apparently healthy lovebirds from 8 flocks in Texas were sampled, including 113 peach-faced lovebirds (Agapornis roseicollis), 32 masked lovebirds (Agapornis personata), and 53 Fischer's lovebirds (Agapornis fischeri). Smears made from cloacal swab samples of individual birds were stained with calcofluor white M2R stain and examined for the presence of spores. Microsporidian spores were identified in the droppings of 25% of the lovebirds sampled. Peach-faced and masked lovebirds were more likely to shed spores than were Fischer's lovebirds (χ2 = 15.905, P < .001). No difference in spore shedding was found between juvenile and adult birds (χ2 = 0.157, P = .692; odds ratio = 1.659; 95% CI, 0.736–3.736). Lovebirds (n = 111) were also assayed for psittacine beak and feather disease virus by polymerase chain reaction (PCR) testing of individual blood samples. Lovebirds that were PCR positive for psittacine beak and feather disease virus (n = 23) were approximately 3 times more likely to shed microsporidian spores than were lovebirds that were PCR negative (χ2 = 3.981, P = .046; odds ratio = 2.6; 95% CI, 1.013–6.754). These results suggest that companion birds without obvious clinical illness commonly shed microsporidian spores. These birds may be a source of spore contamination for opportunistic infections in humans.

EP005 Influence of Caspofungin on blocking Candida adherence to epithelial cells in a thrush model

Fungal keratitis is a common disease that causes blindness. An effective animal model for fungal keratitis is essential for advancing research on this disease. Our objective is to develop a novel mouse model of Fusarium solani keratitis through the inoculation of fluorescent-labeled fungi into the cornea to facilitate the accurate and early identification and screening of fungal infections. F. solani was used as the model fungus in this study. In in vitro experiment, the effects of Calcofluor White (CFW) staining concentration and duration on the fluorescence intensity of F. solani were determined through the mean fluorescence intensity (MFI); the effects of CFW staining on the growth of F. solani were determined by the colony diameter. In in vivo experiment, the F. solani keratitis mice were induced and divided into a CFW-unlabeled and CFW-labeled groups. The positive rate, corneal lesion score and several positive rate determination methods were measured. The MFIs of F. solani in the 30 μg/ml CFW-30 min, 90 μg/ml CFW-10 min and 90 μg/ml CFW-30 min groups were higher than that in the 10 μg/ml CFW-10 min group (P < 0.01). Compared with the 30 μg/ml CFW-30 min group, only the 90 μg/ml CFW-30 min group showed higher MFI (P < 0.05). No significant difference was observed in the colony diameter in the CFW unstained group compared with that in the 10, 30, 90, 270, or 810 μg/ml CFW groups stained for either 10 or 30 min (P > 0.05). No significant differences (P > 0.05) were observed for the positive rate or the corneal lesion scores between the CFW-unlabeled and the CFW-labeled group. On day 1 and 2, the positive rates of the infected corneas in the scraping group were lower than those in the fluorescence microscopy group (P < 0.05). On day 3, these observe methods showed no significant difference (P > 0.05). Thus, these experiments established a novel murine model of F. solani keratitis utilizing fluorescent labeled fungi. This model facilitates the accurate identification and screening of fungal infections during the early stages of fungal keratitis and provides a novel and reliable technology to study the fungal keratitis.

Comparison of diagnostic methods in the evaluation of onychomycosis

Background Onychomycosis is a common problem seen in clinical practice. Given the differential diagnosis of dystrophic nails, it is helpful to obtain a definitive diagnosis of dermatophyte infection before the initiation of antifungal therapy. Potassium hydroxide (KOH) preparation and fungal culture, which are typically used in the diagnosis of these infections, often yield false-negative results. Recent reports have suggested that nail plate biopsy using periodic acid-Schiff (PAS) (Bx/PAS) stain may be a very sensitive technique for the diagnosis of onychomycosis. Objective The purpose of this study was to compare KOH preparation, culture, Bx/PAS stain, and calcofluor white (CW) stain in the diagnosis of onychomycosis and to determine their sensitivity and specificity. Methods We evaluated 105 patients with suspected onychomycosis using 4 diagnostic methods: KOH preparation, culture, Bx/PAS, and CW stain. CW stain binds to cellulose and chitin, and fluoresces when exposed to UV radiation. It is a highly sensitive and specific technique for the detection of dermatophytes. To determine the clinical usefulness and performance characteristics of each test, CW was chosen as the gold standard for statistical analysis. Results Of the patients, 93 had at least 1 of the 4 diagnostic methods positive for the presence of organisms. The following were calculated for each test: sensitivity; specificity; positive predictive value; and negative predictive value. The sensitivities of each of the techniques were as follows: KOH 80%; Bx/PAS 92%; and culture 59%. Both KOH and Bx/PAS methods were more sensitive than culture ( P = .00002). Bx/PAS was also more sensitive than KOH ( P = .03). The specificities were as follows: KOH 72%; Bx/PAS 72%; and culture 82%. The positive predictive value calculated for the different techniques were: KOH 88%; Bx/PAS 89.7%; and culture 90%. In terms of negative predictive value, the results were: KOH 58%; Bx/PAS 77%; and culture 43%. Conclusion Bx/PAS is the most sensitive method for the diagnosis of onychomycosis. It is also superior to the other methods in its negative predictive value. It is indicated if other methods are negative and clinical suspicion is high, and potentially is the single method of choice for the evaluation of onychomycosis.

Phylogenetic analysis identifies the 'megabacterium' of birds as a novel anamorphic ascomycetous yeast, Macrorhabdus ornithogaster gen. nov., sp. nov.

An organism commonly referred to as 'megabacterium' colonizes the gastric isthmus of many species of birds. It is weakly gram-positive and periodic acid-Schiff-positive and stains with silver stains. Previous studies have shown that it has a nucleus and a cell wall similar to those seen in fungi. Calcofluor white M2R staining suggests that the cell wall contains chitin, a eukaryote-specific substance, and rRNA in situ hybridization demonstrates that it is a eukaryote. To characterize this organism phylogenetically, DNA was extracted from purified cells. rDNA was readily amplified by PCR with pan-fungal DNA primer sets and primer sets derived from the newly determined sequence, but not with bacteria-specific primer sets. Specific primer sets amplified rDNA from isthmus scrapings from an infected bird, but not from a non-infected bird or other control DNA. The sequence was confirmed to derive from the purified organism by in situ rRNA hybridization using a specific probe. Phylogenetic analysis of sequences of the 18S rDNA and domain D1/D2 of 26S rDNA showed the organism to be a previously undescribed anamorphic ascomycetous yeast representing a new genus. The name Macrorhabdus ornithogaster gen. nov., sp. nov. is proposed for this organism. The type material is CBS 9251T (= NRRL Y-27487T).

Corneal Neovascularization after Excimer Keratectomy Wounds in Matrilysin-Deficient Mice

Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing. Matrilysin-deficient mice (n = 17) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic factors, the levels of basic fibroblast growth factor (bFGF) were compared with those of vascular endothelial growth factor (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV). The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% +/- 5.2% and 18.7% +/- 5.8% at days 3 and 7, respectively, compared with 5.3% +/- 2.4% and 5.5% +/- 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 +/- 0.55 mm(2) and 0.77 +/- 0.34 mm(2), respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV). Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.

Invasive aspergillosis: results of an 8-year study

We analysed retrospectively 90 cases of invasive aspergillosis (IA) which occurred at the University Hospital and the Thoraxklinik gGmbH Heidelberg between 1991 and 1998. 71 cases were histologically proven, 19 were probable diseases. There were 49 male and 41 female patients, with a mean age of 51.5 years (range 16 days to 80 years). Underlying diseases were: hematological malignancies in 52% (n = 47; 24 with acute leukemia), solid organ transplantation (n = 11; 9 liver, 1 kidney, 1 heart), solid cancer (n = 10), others (n = 21), and in one case no underlying disease was diagnosed. Only 54 cases (60%) were correctly diagnosed as IA during lifetime of the patients. In 59 cases (65%) only the lung was affected, 25 patients suffered from disseminated IA, in 6 patients only extrapulmonary lesions were present. 11 patients underwent lung surgery, 63 patients received antimycotic drugs (44 amphotericin B, 15 fluconazole, 4 itraconazole), 21 were not treated antimycotically. 68 patients (71%) died, from these 30 (36%) due to IA during remission of the underlying disease. The laboratory methods showed the following sensitivities, respectively: microscopy by calcofluor white staining 17%, culture 69%, Aspergillus-PCR from respiratory tract samples and biopsies 95%, galactomannan antigen detection by latex agglutination 28%, by enzyme immunoassay 59%, Aspergillus antibody detection 23%.