US-Japan workshops in medical mycology: past, present and future.

The Extramural Mycology Program of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) has organized and implemented a five workshop series in medical mycology during a critical period in the evolution of contemporary medical mycology (1992 to 2000; http://www.niaid.nih.gov/research/dmid.htm). The goals of the workshop series were to: initiate interactions; build collaborations; identify research needs; turn needs into opportunities; stimulate molecular research in medical mycology; and summarize recommendations emerging from the workshop proceedings. A recurring recommendation in the series was to foster communications within and beyond the field of medical mycology. US-Japan interactions were noted as one specific example of potential information exchange for mutual benefit. The first formal action directed at this recommendation was the workshop Emergence and Recognition of Fungal Diseases convened under the auspices of the US-Japan Cooperative Medical Science Program (USJCMSP; http://www.niaid. nih.gov/dmid/us%5Fjapan/default.htm) in Bethesda, Maryland USA on 30 June 1999 (D.M. Dixon & T. Matsumoto, co-chairs). A major goal of the workshop was to present contemporary medical mycology to the Joint Committee of the USJCMSP through representative research presentations in order to make the Committee aware of current status in the field, and the potential for scientific interactions. The second formal action is the workshop, under the auspices of the Japanese Society for Medical Mycology Medical Perspectives of Fungal Genome Studies scheduled for 28 November 2000 in Tokyo, Japan (T. Matsumoto & D.M. Dixon, co-chairs). The NIAID Mycology Workshop series recommended interactions between the following groups: academic and pharmaceutical; medical and molecular (model systems); medical and plant pathogens; basic and clinical; mycologists and immunologists. The first two US-Japan workshops can be viewed as consistent with these recommendations, and serve as a Western/Eastern gateway for exchange. The focus of the second US-Japan workshop on genome projects for the medically important fungi provides an excellent model for international communications. Given the tsunami of information that is flowing from genomics and bioinformatics, it is clear that global interactions will be essential in managing and interpreting the data.

The physician healer: ancient magic or modern science?

The therapeutic role of general practitioners (GPs) is one that, over the years, has slowly diminished with the growing fashion for evidence-based medicine. However, it is clear that the art of healing and the strength of the doctor-patient relationship play a vital role in improving the well-being of patients. This is exemplified by the placebo effect, where the attitude of the doctor can make an appreciable difference to the psychological response of the patient who feels the need to be understood and listened to empathically. By maximizing the role of the physician healer, there is considerable scope for bridging the gap left by the impersonality of medical science, while at the same time increasing the GP's effectiveness.

Development of vaccines and their use in the prevention of fungal infections.

Vaccine approaches to infectious diseases are widely applied and appreciated. Disciplines such as bacteriology and virology have a rich history of successful vaccine development. The complexity of eukaryotic systems presents additional challenges to the development of vaccines against them. These challenges are being met in the fields of parasitology, and are being revisited for application in oncology. Vaccine opportunities exist in medical mycology. The National Institute of Allergy and Infectious Diseases has held a series of workshops in medical mycology where the need to develop vaccines for fungal diseases was noted and where important opportunities were discussed. Major advances in vaccinology and the technology of antigen preparation and delivery have increased feasibility and heightened interest. The recent epidemic of coccidioidomycosis in the American Southwest has demonstrated the need for developing a vaccine as an effective preventive measure for those living in and for those who subsequently move into regions with the endemic mycoses. The XIIth Congress of the International Society for Human and Animal Mycology included a symposium that summarized new vaccination strategies for selected fungi: Candida albicans, Coccidioides immitis, and Trichophyton verrucosum. The goal of the present summary is to provide representative examples of continuing efforts relating to vaccine development within the medical mycological community highlighting Blastomyces dermatidis, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Pythiumn insidiosum.

A comparison of gait with solid, dynamic, and no ankle-foot orthoses in children with spastic cerebral palsy.

BACKGROUND AND PURPOSE: This study compared the effects of dynamic ankle-foot orthoses (DAFOs) with a plantar-flexion stop, polypropylene solid ankle-foot orthoses (AFOs), and no AFOs on the gait of children with cerebral palsy (CP). These orthoses were used to reduce the excessive ankle plantar flexion during the stance phase of gait. SUBJECTS AND METHODS: Ten children with spastic CP (6 with diplegia and 4 with hemiplegia) were tested after wearing no AFOs for an initial 2-week period, solid AFOs for 1 month, no AFOs for an additional 2 weeks, and DAFOs for 1 month. The effects of the two orthoses and no AFOs on lower-extremity muscle timing, joint motions, and temporal-distance characteristics were compared. RESULTS: Both orthoses increased stride length, decreased cadence, and reduced excessive ankle plantar flexion when compared with no orthoses. No differences were found for the gait variables when comparing the two orthoses. CONCLUSION AND DISCUSSION: Based on the data, the authors believe that although both orthoses would be recommended for children with spastic CP and excessive ankle plantar flexion during stance, additional individual factors should be considered when selecting either orthosis.

Fungal infections: a growing threat.

THE EMERGENCE OF newly identified fungal pathogens and the reemergence of previously uncommon fungal diseases is primarily related to increases in the numbers of susceptible persons: people with HIV infection, bone marrow and organ transplant recipients, cancer patients being treated with chemotherapy, critically ill persons, and very low birth weight ( < or = 1500 g) infants. These immunocompromised populations are at risk for infection not only with opportunistic pathogens (for example, Pneumocystis, Candida, Cryptococcus, Trichosporon, Malassezia, Aspergillus, Penicillium marneffei, and numerous other moulds or yeasts) but also with fungal pathogens that usually infect otherwise healthy persons not previously exposed to endemic fungi (for example, Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis) and Sporothrix schenckii. Morbidity, mortality, and health care costs associated with fungal infections are high. Addressing the emergence of fungal diseases will require increased surveillance coupled with the availability of rapid, noninvasive diagnostic tests; monitoring the development of resistance to antifungal agents; and research focused on the understanding, prevention, and control of fungal infections.

GM-CSF is required for the Pseudomonas exotoxin A-induced proliferation of immature T cells in athymic mice.

Previous studies have demonstrated that Pseudomonas exotoxin A stimulated the proliferation of immature T lymphocytes within the splenocytes of athymic mice. These studies were performed to determine which lymphokines were involved in the proliferation of the immature T cells. The results of this study indicate that exotoxin A does not induce the production of interleukin-2 or tumor necrosis factor from B cell-depleted splenotypes from athymic mice. However, exotoxin A does induce the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from B cell-depleted splenocytes. Furthermore, the GM-CSF was shown to be produced by a Thy1+, CD4-, CD8- T lymphocyte. The addition of anti-GM-CSF antibody abrogates the exotoxin A-induced proliferation of B cell-depleted splenocytes from athymic mice. Thus, these data indicate that exotoxin A induces the production of GM-CSF from immature T lymphocytes within the splenocytes of athymic mice and the exotoxin A-induced proliferation of these immature T cells is dependent on the presence of GM-CSF.

Proliferation of immature T cells within the splenocytes of athymic mice by Pseudomonas exotoxin A.

Pseudomonas aeruginosa exotoxin A has been shown to stimulate splenocytes from athymic nude mice. The present studies were performed to characterize the exotoxin A-responsive cells within the splenocytes of athymic nude mice. The results of these studies indicate that exotoxin A-responsive cells were represented in the nylon wool-adherent cell population and in the Ig-depleted splenocyte population (IgNA). Flow microfluorimetry analysis indicated that exotoxin A induced the expansion of Thy1+, CD3-, CD4-, and CD8- cells which also expressed the heat-stable antigen (HSA), an antigen expressed on immature T lymphocytes. Depletion of Thy1+ cells abrogated the exotoxin A-induced proliferation; however, depletion of CD4+ and CD8+ cells did not affect the exotoxin A-induced proliferation of IgNA cells. Thus, the results indicate that exotoxin A stimulates the proliferation of immature T cells within the splenocytes of nude mice that are Thy1+, HSA+, CD3-, CD4-, CD8-.

The Ca-activated chloride channel of Ascaris suum conducts volatile fatty acids produced by anaerobic respiration: a patch-clamp study.

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of carboxylic acids, a number of which are products of anaerobic respiration, was investigated using the patch-clamp technique and isolated inside-out patches of muscle membrane. The channel has a conductance of 140 pS in symmetrical 140 mM chloride. Replacement of internal chloride with various carboxylic acids (140 mM) caused large hyperpolarizing shifts in the reversal potential. Permeability ratios, relative to chloride, were calculated for each acid. The relationship between permeability ratio and ionic size is inverse and linear predicting a pore diameter of 6.55 A. This simple relationship was not observed between ionic size and conductance. Calculation of the transition state energy required to transfer a single methyl group from aqueous phase to the binding site afforded a value that was low but favorable, indicating a cationic binding site of low field strength. As the channel is able to open fully at the resting membrane potential of Ascaris and is permeable to fatty acids produced by anaerobic respiration, the possible role of this channel in the removal of metabolic products across the muscle membrane is discussed.

Relationship between Candida albicans epidermolytic proteinase activity and virulence in mice.

Five strains of Candida albicans with previously characterized epidermolytic acid proteinase activity were evaluated for virulence following intravenous (i.v.) injection in mice. Increased proteinase activity was associated with increased virulence in female, NYLAR mice receiving 10(6) cells i.v. Mean mortality times (1.25, 2.0, 2.0, 4.25 and 19.6 days, in groups of 20 mice for each of the five strains) correlated directly with degree of proteinase activity. Three of the strains were selected for additional in vivo study and the association between increased proteinase activity and increased mortality rates was confirmed in dose-response studies in two additional strains of mice. The mean survival times appeared to be independent of fungal growth rate in vitro. These results support the positive correlation between proteinase activity and virulence.

Conversion of dynorphin A(1-8) to [Leu5]-enkephalin in rat central nervous tissue during development.

Rat central nervous tissue contains enzymic activity that is able to convert the kappa-receptor preferring opioid peptide dynorphin A(1-8) to the delta-nu-receptor preferring opioid peptide [Leu5]enkephalin. The ontogeny of this conversion process has been studied in vitro using cortex, striatum, cerebellum and spinal cord tissues of the developing rat brain. Evidence for the enzymic cleavage of the Leu5-Arg6 bond of dynorphin A(1-8) to afford [Leu5]enkephalin was observed as early as neonatal day 1. The degree of conversion increased up to day 7, at which time adult levels were attained. Results in all tissues studied were similar. The relationship between the increase in the conversion process with age and the ontogeny of opioid peptides and their receptors may indicate an important role for the process in the developing nervous system.

A phaeohyphomycotic cyst and peritonitis caused by Phialemonium species and a reevaluation of its taxonomy.

Two cases of human fungal infections caused by members of the genus Phialemonium, a genus proposed by Gams and McGinnis (1983) for fungi intermediate between the genera Acremonium and Phialophora, are presented. The first case was a phaeohyphomycotic cyst on the foot of a renal transplant recipient. The fungus was detected by direct examination and histopathology and was recovered by several procedures over 4 months. It was flat, glabrous, and white becoming yellow with the production of a diffusible yellow pigment; it had conidiophores that were mostly solitary and lateral and terminal phialides and adelophialides with distinct collarettes producing cylindrical to curved conidia. The isolate resembled both Phialemonium dimorphosporum and Phialemonium curvatum, although its characteristics were more consistent with those of the latter. The second case was peritonitis in a renal transplant recipient. The fungus was white-to-cream colored and yeast like, but later became black with a green diffusible pigment, and produced obovoid conidia; it was easily identified as Phialemonium obovatum. Difficulties encountered in the identification and taxonomy of members of this genus highlight the need for standardized conditions, e.g., potato dextrose agar culture incubated at 24 to 25 degrees C for morphologic comparisons, to control significant variations due to culture conditions.

A patch-clamp study of the ionic selectivity of the large conductance, Ca-activated chloride channel in muscle vesicles prepared from Ascaris suum.

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of anions has been investigated using the patch-clamp technique on isolated inside-out patches of muscle membrane. Symmetrical Cl solutions (140 mM) produced single-channel I/V plots with reversal potentials of 0 mV, substitution of bath Cl by 140 mM NO3, Br and I caused depolarizing shifts in the reversal potentials. Replacement of the internal Cl by F (140 mM) caused a large hyperpolarizing shift in the reversal potential. The channel displayed a permeability sequence of I > Br = NO3 > Cl > F which differed from the corresponding conductance sequence Cl > NO3 = Br = I > F. The ionic environment within the channel pore has been investigated using Reuter and Stevens (1980) plots to describe the selectivity and "fluidity" of the channel pore. In addition, the approach of Wright and Diamond (1977) was employed to estimate the number of cationic binding sites within the channel pore. The channel is relatively fluid but the number of cationic binding sites varies inversely with the ionic radius of the anion from 2.15 for F to 0.89 for the large planar anion NO3.

Human infectious corneal ulcer caused by Pythium insidiosum.

Pythium insidiosum is a fungus-like organism known to infect a variety of animals. In humans, the few known cases involving Pythium have included arterial infections and cellulitis. We present what we believe to be the first case of P. insidiosum recovered from a human corneal ulcer. The organism is difficult to isolate, causing delays in diagnosis. It is also resistant to the usual antifungal medications, making surgical excision the treatment of choice.

Patch-clamp and chloride channels in Ascaris suum.

Single-channel electrophysiology is an invaluable tool fo the study of ion channels. However, it is a technique that has failed to attract widespread use by parasitologists. Here, Diane Dixon and Richard Martin outline the principles undelrying single channel recording and highlight its uses in the discovery of a new and unusual chloride channel in the musculature of Ascaris suum.

Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.

A multistate outbreak of sporotrichosis associated with sphagnum moss.

In the spring of 1988, the largest documented US outbreak of cutaneous sporotrichosis to date occurred, with 84 cases among persons from 15 states who were exposed to Wisconsin-grown sphagnum moss used in packing evergreen tree seedlings. In New York State, 13 cases occurred among 109 forestry workers. All 13 cases occurred among 76 workers who had handled evergreen seedlings and moss (attack rate = 17%). For those exposed to evergreens and moss, the risk of infection increased as worktime exposure to moss increased (attack rates: less than 10 hours, 8%; 10-19 hours, 12%; greater than 19 hours, 33%). While environmental samples of moss from the Wisconsin supplier were negative, Sporothrix schenckii was cultured from multiple samples of the sphagnum moss obtained from one of six Pennsylvania tree nurseries, representing the nursery that was identified as the source for 79 (94%) of the moss-associated cases. Differences in tree-handling procedures at this nursery--including the use of 1- to 3-year-old moss to pack seedlings, use of a pond water source to wet the moss, use of an organic polymer gel on the seedling root system, and underground storage and longer storage of moss-packed seedlings before shipping--suggested possible explanations for the association. Efforts to prevent sporotrichosis among persons handling evergreen seedlings should include the use of alternate types of packing material (e.g., cedar wood chips or shredded paper) and protective clothing such as gloves and long-sleeved shirts.

DNA typing of isolates associated with the 1988 sporotrichosis epidemic.

DNA typing techniques were used to examine selected clinical and environmental isolates of Sporothorix spp. recovered from the 1988 sporotrichosis epidemic in multiple states of the United States. Previous studies indicated that isolates in one of the six morphologically or physiologically distinct groups (group I) obtained from environmental sources were Sporothrix schenckii and were the possible etiologic agents responsible for the epidemic. To assess this hypothesis at the genetic level, whole-cell DNA was extracted from selected clinical isolates and representative members of each of the six environmental groups subjected to endonuclease digestion and then analyzed by gel electrophresis. DNA types were assigned on the basis of restriction fragment length polymorphism patterns. One DNA type was common to clinical and group I isolates but was dissimilar from the DNA types exhibited by groups II to VI. In contrast, a variety of DNA types were associated with isolates in groups II to VI. The latter groups appeared to make up a heterogeneous collection of fungi, with some members of the same group having different DNA types but with others from different groups possessing identical DNA types. Thus, DNA typing studies confirmed that group I environmental isolates are indistinguishable from clinical isolates and that group II to VI isolates represent a complex of related fungi with similar Sporothrix anamorphs.