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In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis. Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), single nucleotide polymorphisms, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR)-RFLP and microsatellites. By means of these methods, several important biological questions related to speciation, mode of reproduction and population genetics could be approached. The basic information obtained from this approach has implications in the understanding of these pathogenic fungi in relation to their behavior and the development of pathogenic features, such as resistance to antimicrobials and virulence factors used for colonization of mammalian hosts. The knowledge obtained from these studies could also be used for the development of innovative diagnostic methods, as well as for novel therapeutic approaches and production of vaccines.

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG) under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU). Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.

Lewis rat alveolar macrophages (AM) were harvested and exposed in vitro to Cryptococcus neoformans to investigate the induction of inflammatory cytokines. AM in tissue culture wells were incubated with viable yeast cells of C. neoformans or the capsular polysaccharide, glucuronoxylomannan (GXM), with or without rabbit anti-C. neoformans antiserum. At 3, 6, 12 and 24 h, AM were washed, lyzed and total RNA was isolated. Using reverse transcription-PCR, the transcripts of cytokine genes were semi-quantified by comparison with constitutive transcripts. Incubation of AM with lipopolysaccharide, as positive control, induced elevated levels of the three transcripts measured: interleukin (IL)-1 alpha, IL-6 and tumour necrosis factor (TNF)-alpha. Under the same conditions, no obvious changes were observed in the levels of transcription of these cytokines by AM after exposure to several strains of C. neoformans. However, AM that were incubated with both the yeast cells and rabbit polyclonal antisera to C. neoformans manifested significantly increased levels of mRNA for IL-6, but not IL-1 alpha or TNF-alpha. This increased level of IL-6 mRNA was detectable after incubation for 6 or 12 h. Levels of transcription in AM were unaffected by exposure to normal rabbit serum, specific antiserum alone. GXM at concentrations of 10, 100 or 500 micrograms ml-1, or GXM and antiserum. Adsorption of the antiserum with heat-killed yeast cells of C. neoformans diminished its ability to induce IL-6 mRNA in combination with fresh, viable yeast cells. The induction of IL-6 mRNA by yeast cells and antiserum does not require intact complement. In the absence of complement, the rabbit antiserum served as a potent opsonin and markedly increased phagocytosis of C. neoformans by AM. These results indicate that antibody-opsonized C. neoformans are readily phagocytosed by rat AM, and that antibody-mediated phagocytosis may differ from complement-mediated phagocytosis in the subsequent stimulation of IL-6.

Nucleotide sequences obtained from regions of the ribosomal DNA repeat were compared by phylogenetic methods and combined with a statistical evaluation to clarify the relationships among the genera Cystofilobasidium, Filobasidium (F.) and Filobasidiella (Fl.), to assess the affinity of Filobasidiella neoformans and Filobasidiella depauperata, and to compare the varieties of Fl. neoformans. With appropriate primers, the nuclear 18S, 5.8S and internal transcribed spacer (ITS) regions of the ribosomal RNA genes (rDNA) of 10 strains were amplified with the polymerase chain reaction. The resulting DNA products were compared by digestion with endonucleases and analysis of restriction fragments. Single strands of the 5.8S rDNA and ITS regions were subsequently sequenced by the dideoxy method. Statistical support for the phylogeny inferred from parsimony analysis of aligned 5.8S rDNA sequences was determined by bootstrapping. There were no nucleotide substitutions in this region, nor in the ITS, among strains of Fl. neoformans that differ in variety and serotype. There was strong support for retaining Fl. depauperata and Fl. neoformans in the same genus, but nucleotide substitutions can be used to distinguish the two species. There was no support for combining the genera Filobasidium, Filobasidiella or Cystofilobasidium.

Summary: In experiments with a surface film test it was found that variation of certain test conditions affected the relative activity of two detergent-sterilizer formulations. It was also found that to achieve a high degree of reproducibility in the residual count on a treated surface from one occasion to another, it is necessary to control closely the numbers of bacteria present in the dried milk film.

An annotated list of 210 corrections and emendations relative to the location (in recognized culture collections) of type strains, neotype strains and reference strains of bacteria as proposed by Sneath and Skerman (1966, 1).

The addition of [51Cr]-labeled yeast cells of Cryptococcus neoformans to monolayers of Lewis rat alveolar macrophages (AM phi) provided a sensitive and reproducible in vitro assay of phagocytosis. AM phi and yeast cells were incubated in 10% (v:v) normal rat serum for 1 h, non-AM phi associated yeast cells were removed and the AM phi-associated radioactivity (phagocytosis) determined. Replicate wells were replenished with fresh medium and reincubated. At different times, yeast-AM phi monolayers were treated with a non-cryptococcocidal mixture of DNAse and sodium deoxycholate to release the yeast cells from the AM phi. The fate of the yeast cells was critically evaluated by [51Cr]-release and viable plate counts. Killing was detected by plate counts within an hour following phagocytosis and did not increase significantly during the next 5 h. Strains of C. neoformans with small, medium, or large capsules varied in their susceptibility to killing from 10% to 95% but susceptibility to killing was not directly related to capsule size and the extent of phagocytosis. Release of 51Cr did not correlate with viability as determined by culture. The 51Cr was associated with two pools in the yeast cells; one, representing 15-20% of the radiolabel, was easily released and was probably bound to low molecular weight compounds in the cytoplasm. The majority of label was tightly bound to the particulate alkali-soluble cell wall fraction.

An in vitro method for the assessment of neutrophil granulocyte chemotaxis employing a cytocentrifuge has been evaluated. The cytocentrifuge is used to directly sediment leukocytes on a Millipore filter, and thus (1) eliminates errors due to abnormal or unequal settling of the cells and (2) eliminates the need for serum, plasma, or albumin on the cell side of the chemotactic chamber to obtain optimal chemotaxis. In addition, other advantages of the method are that it requires smaller volumes of blood, less handling of the cells, and less time is required for visual counting of the neutrophils. The method has been utilized to assess chemotactic activity of neutrophils from 80 control subjects and over 100 patients with various diseases and to study the effects of various pharmacologic agents on neutrophil chemotactic responsiveness.

Regional cerebral blood flow has been evaluated by recording the first passage of an intravenous bolus of sodium pertechnetate through the cerebral circulation with a scintillation camera. In preliminary studies, interpretation was based either on visual assessment of serial 2-sec scintillation camera images or on analysis of regional time-activity curves. Abnormalities were found in 57% of patients with clinical evidence of stroke, in 74% of those with arteriographic abnormalities, and in 92% with severe carotid stenosis (2, 3).

A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.

Austin-Moore prostheses with a porous surface have been used recently to improve stabilization in total joint arthroplasty. However, these prostheses expose tissue directly to greater metallic surface area than previous nonporous designs. Cobalt levels were measured in the urine of patients who had received porous and nonporous prostheses, to assess any increased risk of metal toxicity posed by the porous design. Although a higher mean urinary cobalt concentration was found for patients with porous prostheses, the absolute levels were comparable with those of patients with nonporous prostheses. The prostheses with a porous surface appear to pose no significant added risk of metal toxicity as compared with the nonporous design.

Platelets from nine normal male subjects were labeled with In-111 8-hydroxyquinoline (In-111 oxine) in the presence of plasma in either "closed" blood transfer packs or in "open" test tubes. The mean labeling efficiencies in these two systems were 27 and 53%, respectively. Mean survival time of In-111-labeled autologous platelets was 8.76 days, with a standard deviation of 1.05 according to the maximum-likelihood estimate of the gamma-function model. The initial recovery of In-111 platelets in the circulation was 57% with a standard deviation of 11%. The distribution of In-111 platelets in liver and spleen was quantitated by anterior, posterior, and transmission gamma-camera imaging. During the first 30 min, 38% of the injected dose accumulated in the spleen, 13% in the liver. No significant increase in In-111 radioactivity was observed in either of the two organs over a 3-9-day period. The bone marrow was an additional site of In-111 accumulation. The spleen was the critical organ with respect to radiation dose. The splenic dose was estimated to be 34 rad/mCi In-111 platelets, that of the liver 2.1 rad/mCi. With the injection of 100-150 microCi of In-111-labeled platelets in normal subjects, giving a splenic radiation of 5 rad, a complete 10-day survival study can be performed and uptake of In-111 in different organs can be measured quantitatively for at least 3-4 days.

A radiolabeling method was developed to investigate the interaction in vitro between Cryptococcus neoformans and Lewis rat alveolar macrophages (AM phi). AM phi were harvested by lung lavage, monolayers of adherent cells were established in wells of microtiter plates and [51Cr]-labeled yeast cells were added to the monolayers. After removal of extracellular yeasts, the adherent radioactivity associated with the AM phi was directly proportional both to the number of yeasts added and to the number of yeasts per AM phi as determined by microscopic examination of Giemsa-stained monolayers. Phagocytosis (attachment and/or ingestion) of radiolabeled C. neoformans by AM phi was a sensitive, quantitative and reproducible assay for the evaluation of the AM phi-C. neoformans interaction. AM phi were able to phagocytose encapsulated strains of C. neoformans. The extent of phagocytosis was inversely related to the capsule size. Normal rat serum (NRS) was an excellent source of opsonins for the ingestion. Inactivation of serum complement or depletion of C3 by affinity chromatography removed most of the opsonic activity of NRS. Specific antibodies against C. neoformans did not increase phagocytosis.