Procedure for Fluorescent-Antibody Staining of Virus-Infected Cell Cultures in Plastic Plates

Acetone fixation and fluorescent-antibody staining of virus-infected cell cultures were performed in plastic plates. Proper addition of acetone as a fixative did not alter the plastic.     

Facial expression recognition by people with mobius syndrome

We present an investigation of facial expression recognition by three people (BC, LP, and NC) with Mobius syndrome, a congenital disorder producing facial paralysis. The participants were asked to identify the emotion displayed in 10 examples of facial expressions associated with each of 6 basic emotions from the Ekman and Friesen (1976) series. None of the three people with Mobius syndrome was significantly impaired on this task. On a second test of facial expression recognition using computer-morphed facial expressions, NC showed a statistically significant impairment, BC a borderline deficit, and LP was unimpaired. However, even when impairments were found, people with Mobius syndrome still recognised many of the facial expressions shown to them. The recognition of facial expressions by people who have never been able to produce such signals on their own faces demonstrates that the ability to produce facial expressions is not a necessary prerequisite of their recognition.     

Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis.

The membranous spherule outer wall (SOW) isolated from liquid cultures of Coccidioides immitis has been shown to elicit reactivity with human anti-Coccidioides antibody by immunofluorescence and the immunodiffusion-tube precipitin assay. The serologically reactive components were extracted from SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG). The OG-soluble fraction of SOW was shown to be reactive with immunoglobulin G in 25 serum samples from coccidioidomycosis patients by an enzyme-linked immunosorbent assay. The isolated SOW and OG-soluble fraction of SOW were also demonstrated to be capable of eliciting lymphocyte blastogenesis. The antigenic and protein compositions of the OG-soluble fraction were examined by two-dimensional immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two antigens which were extracted from SOW were identified as antigens 2 and CS on the basis of the coccidioidin-anticoccidioidin reference system. The latter was isolated earlier and shown to correspond to a molecular mass (Mr) of 19 X 10(3) by SDS-PAGE under reducing conditions. This same electrophoresis band was shown to be reactive with sera from coccidioidomycosis patients by immunoblot analysis. One other SDS-PAGE component of the OG-soluble fraction of SOW with an Mr of 66 X 10(3) was shown to be reactive with sera from patients by immunoblot analysis. The SOW of C. immitis represents an important reservoir of immunoreactive wall components which has not previously been reported.     

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.     

Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis.

Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.     

The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.     

A vaccine against coccidioidomycosis is justified and attainable.

Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals. Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis. In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.     

I-E/I-C region-associated induction of murine gamma interferon by a haplotype-restricted polyclonal T-cell mitogen derived from Mycoplasma arthritidis

Cell-free supernatants from cultures of Mycoplasma arthritidis induced significant levels of interferon when cocultured with murine splenic cells. On the basis of physicochemical characteristics and antibody neutralization studies, the antiviral substance was identified as gamma interferon. Use of inbred and congenic mouse strains established that splenic cells from mice expressing the H2k and H2d haplotypes produced interferon in response to M. arthritidis culture supernatants, but those from mice with H2b and H2q haplotypes did not. Further studies with recombinant mouse strains established that interferon induction by the mycoplasma supernatant was associated with the haplotype expressed at the I-E/I-C subregion of the murine major histocompatibility complex. The specificity seen for interferon induction was identical with that reported earlier for induction of cytotoxic lymphocytes and for lymphocyte proliferation in response to the mitogen. All of these reactions appear to be dependent upon binding of the mitogen to specific I-E/I-C region-coded products present on splenic cell surfaces. The observations presented introduce the concept that microbial mitogens or their lymphokine products might modify immune responses and defense mechanisms of the naive host in a genetically restricted manner.