A novel vaccine platform using glucan particles for induction of protective responses against Francisella tularensis and other pathogens.

Vaccines are considered the bedrock of preventive medicine. However, for many pathogens, it has been challenging to develop vaccines that stimulate protective, long-lasting immunity. We have developed a novel approach using ß-1,3-D-glucans (BGs), natural polysaccharides abundantly present in fungal cell walls, as a biomaterial platform for vaccine delivery. BGs simultaneously provide for receptor-targeted antigen delivery to specialized antigen-presenting cells together with adjuvant properties to stimulate antigen-specific and trained non-specific immune responses. This review focuses on various approaches of using BG particles (GPs) to develop bacterial and fungal vaccine candidates. A special case history for the development of an effective GP tularaemia vaccine candidate is highlighted.

Phenotypic and functional characterization of human lymphocytes activated by interleukin-2 to directly inhibit growth of Cryptococcus neoformans in vitro.

Recently we demonstrated that the nonadherent (to plastic) fraction of human PBMC could be activated by IL-2 to inhibit Cryptococcus neoformans growth. Here we characterize the antifungal effector cells. Depletion by panning of natural killer (NK) (CD16+, CD56+) cells from nylon wool-treated, IL-2-activated PBMC markedly decreased lytic activity against a tumor cell target (K562) but did not affect antifungal activity. Panning out T (CD3+, CD5+) cells enhanced activity against tumor cells but partially abrogated activity against C. neoformans. IL-2-activated T cells of 95% purity, obtained by panning out NK cells from PBMC forming rosettes with sheep erythrocytes, had excellent antifungal activity but suboptimal antitumor activity. The nonrosetted cells (which were virtually free of T cells and enriched for NK cells) had both antitumor and antifungal activity, even if cultured without IL-2. CD4+, CD8+, and CD56+ cells, purified by positive selection by panning, directly inhibited cryptococcal growth. Conjugate formation between fungi and both CD56+ and CD5+ effector cells was demonstrated by videomicroscopy and immunoperoxidase staining. Thus, IL-2-activated T cells and NK cells form conjugates with and directly inhibit the growth of C. neoformans. To our knowledge, these data are the first demonstration of human T cells directly inhibiting growth of a microbial target.

Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin.

We studied the receptors on human cultured macrophages (MO-M phi) responsible for binding encapsulated and isogenic mutant acapsular strains of Cryptococcus neoformans, and whether such binding leads to a phagocytic event. Both strains required opsonization with complement components in normal human serum in order for binding to occur. Binding of the acapsular, but not the encapsulated, strain led to phagocytosis. MAb directed against any of the three defined complement receptors (CR) on MO-M phi (CR1, CR3, and CR4) profoundly inhibited binding of serum-opsonized encapsulated (and to a lesser extent acapsular) organisms to MO-M phi. Immunofluorescence studies demonstrated migration of CR to the area of the cryptococcal binding site. Trypsin and elastase inhibited binding of encapsulated and, to a lesser extent, acapsular yeasts to MO-M phi. Binding of encapsulated C. neoformans was profoundly inhibited by incubation in the cold or by inhibitors of receptor capping and actin microfilaments. Thus, multiple CR appear to contribute to binding of serum-opsonized encapsulated C. neoformans by MO-M phi. Binding is an energy-dependent process that requires conformational changes in actin yet does not lead to phagocytosis of the organism. In contrast, energy is not required for binding of acapsular yeasts by MO-M phi and binding triggers phagocytosis.

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation.

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.

Fungal Recognition and Host Defense Mechanisms.

Fungi have emerged as premier opportunistic microbes of the 21st century, having a considerable impact on human morbidity and mortality. The huge increase in incidence of these diseases is largely due to the HIV pandemic and use of immunosuppressive therapies, underscoring the importance of the immune system in defense against fungi. This article will address how the mammalian immune system recognizes and mounts a defense against medically relevant fungal species.

Chloroquine and the fungal phagosome.

The antimalarial drug chloroquine accumulates inside the macrophage phagolysosome by ion trapping where it exerts potent antifungal activity against Histoplasma capsulatum and Cryptococcus neoformans by distinct mechanisms. Chloroquine inhibits growth of H. capsulatum by pH-dependent iron deprivation, whereas it is directly toxic to C. neoformans. Clearly, clinical studies are required to document the potential therapeutic efficacy of chloroquine or related congeners as adjuvant therapy in fungal disease. Moreover, the diversity of pathogenic microorganisms inhibited and/or killed by chloroquine makes this drug an attractive candidate for prophylactic therapy.

Heterozygous germline ATM mutations do not contribute to radiation-associated malignancies after Hodgkin's disease.

PURPOSE: The successful treatment of Hodgkin's disease has been associated with an increased incidence of secondary malignancies. To investigate whether genetic factors contribute to the development of secondary tumors, we collected family cancer histories and performed mutational analysis of the ataxia-telangiectasia (AT) gene, ATM, in a cohort of Hodgkin's disease survivors with secondary malignancies. ATM was chosen for evaluation because of the increased radiosensitivity of cells derived from AT patients and obligate heterozygotes and the epidemiologic observation that AT carriers are at increased risk for radiation-induced breast cancer. PATIENTS AND METHODS: Fifty-two patients who developed one or more neoplasms after treatment for Hodgkin's disease participated in this study. Personal and family histories of cancer were obtained through patient interviews and review of medical records. ATM mutational analysis was performed using a yeast-based protein truncation assay. RESULTS: Seventy-six secondary neoplasms were observed in this cohort of 52 Hodgkin's disease survivors, with 18 patients (35%) developing more than one secondary neoplasm. Positive family histories of cancer were present in 11 (21%) of 52 patients, compared with three (4%) of 68 Hodgkin's disease patients in a comparison cohort who did not develop secondary neoplasms (P =.008; Fisher's exact test). No germline ATM mutations were identified, resulting in an estimated AT carrier frequency in this population of 0% (90% confidence interval, 0% to 4%). CONCLUSION: Analysis of the number of tumors per individual and the family history of cancer in our cohort suggests that genetic factors may contribute to development of secondary neoplasms in a subset of Hodgkin's disease survivors. Mutational analysis, however, does not support a significant role for heterozygous truncating ATM mutations. Future studies evaluating other genes involved in DNA damage response pathways are warranted.

Human neutrophil degranulation stimulated by Aspergillus fumigatus.

Previous studies have established that human neutrophils (PMN) are unable to kill resting conidia (RC) of Aspergillus fumigatus but can kill conidia that have been preincubated in culture medium until swollen but not yet germinated. Compared with swollen conidia (SC), RC stimulate a relatively weak PMN respiratory burst. In the present study, we further examined the mechanisms of resistance of RC to neutrophil killing by comparing neutrophil degranulation and phagocytosis following stimulation by RC and SC opsonized in pooled human serum. RC, compared with SC, stimulated significantly less release of both the primary granule marker beta-glucuronidase and the secondary granule marker lactoferrin. PMN also phagocytosed significantly greater numbers of SC, although the differences in phagocytosis were not great enough to account for the differences in degranulation. Suboptimal stimulation of degranulation and phagocytosis may thus contribute to the inability of neutrophils to kill RC. Moreover, reagent lactoferrin bound avidly to both RC and SC, raising the possibility that PMN-released lactoferrin may contribute to antifungal activity at the conidial surface by competing for iron or catalyzing the formation of oxygen radicals.

A rapid fluorescent assay to distinguish attached from phagocytized yeast particles.

In studies of phagocytosis and its consequences for cell activation, it is important to distinguish those particulate stimuli which are completely ingested and internalized from those which are only attached to phagocytic surfaces. Ingestion can be profoundly influenced by both the type of opsonins on the surface of the stimulus and the expression and activation of receptors on the phagocytes for these opsonins. We report a new fluorescent assay which facilitates rapid and reproducible quantitation of attached versus fully internalized live or dead yeast particles by phagocytes. The assay employs the fluorescent dye diaethanol (Uvitex 2B) which stains chitin on the cell wall of fungi and is excluded from live phagocytes. The diaethanol assay and a standard, previously published methylene blue dye exclusion assay yielded comparable results using human neutrophils or monocytes incubated with heat-killed, serum-opsonized Candida albicans. The diaethanol assay proved useful in distinguishing differences in effects of various opsonins, as human neutrophils selectively opsonized with either pooled human serum (PHS), IgG (heat-inactivated PHS) or complement (IgG-depleted PHS) completely internalized 69.5%, 91.3% and 52.5% of cell-associated zymosan particles respectively. Finally, the new assay permitted comparisons of differing macrophage populations, as resident murine peritoneal macrophages internalized only 10.6% of complement-opsonized, cell-associated zymosan particles compared with 41.7% when the macrophages were elicited with thioglycolate. The assay should prove useful to investigators studying both fungal phagocytosis and killing, as well as to those performing general studies of receptor expression, regulation and function.

Factitious dermatosis masquerading as recurrent herpes zoster.

A 35-year-old nurse's aide presented with monthly episodes, during her menses, of self-induced cutaneous lesions intended to simulate recurrent herpes zoster. Features of the clinical presentation that prompted the correct diagnosis are discussed.

Radioresistance of culture-induced augmented natural killer-like activity.

Human NK activity is radiosensitive under the control of X-linked genes. We have evaluated the expression of these genes in other forms of cellular cytotoxicity. The NK radioresistant and radiosensitive phenotype is expressed in ADCC. Specific cellular cytotoxicity, generated in a MLC with a radiosensitive donor as responder, was radioresistant. NK-like activity recruited from nonadherent cells of radiosensitive subjects stimulated with allogenic cells, mitogens (PHA, Con A or PWM), or recall antigens (TT or PPD) was radioresistant. The acquisition of radioresistance was relatively rapid, beginning within 24 hr after exposure to PHA, prior to detectable proliferation. Radioresistance of MLR augmented NK-like activity was maximal 3 days after initiation of the culture. MLR augmented NK-like activity was spared by the immunosuppressive polypeptide antibiotic CsA at doses up to 1 micrometer/ml. CsA did, however, reduce acquisition of radioresistance by the NK-like activity at doses above 0.01 mu gm/ml, a concentration which does not inhibit uptake of 3H-thymidine but does reduce the level of specific CML. These data suggest that mitogens and antigens, including allogeneic cells, are recruiting radioresistant NK-like activity which can be distinguished from the radiosensitive spontaneous NK activity of the cell donor. Further, in the MLR, both radiosensitive and radioresistant NK-like activity may be recruited.

Aspergillosis.

The wide spectrum of disease states caused by fungi of the genus Aspergillus is dependent in large measure on environmental conditions leading to exposure, and to local and systemic host defenses. Allergic aspergillosis occurs in individuals hypersensitive to the fungus and is treated by immunosuppression, whereas invasive aspergillosis predominates in the severely immunocompromised and is treated with antifungal therapy. Aspergillomas generally arise from saprophytic colonization of a pre-existing pulmonary cavity with Aspergillus, and may be complicated by life-threatening hemoptosis.

Natural history of the rearfoot angle: preliminary values in 150 children.

The degree of valgus in the heel during weightbearing (the rearfoot angle) is commonly used as an angular criterion in the evaluation and treatment of flatfoot in children and adults. The rearfoot angle has been frequently reported to reduce with age to a vertical heel position attained at approximately 7 years old. The present study evaluated the rearfoot angle in 150 healthy children (age range, 6-16 years). The average rearfoot angle for all children in this study was 4 degrees of valgus (SD, 1.1 degrees; range, 0 degree - 9 degrees of valgus). There was no significant difference in the rearfoot angle in children of different ages. The rearfoot angle did not reduce to the vertical heel position by age 7 as had been reported previously.

Tuberculosis of the foot. A diagnostic challenge.

Two cases of tuberculosis of the foot are described. The diagnosis for each was delayed and unexpected. The clinical features of osteoarticular tuberculosis are reviewed along with practical guidelines for diagnosis.

Reappraisal of the negative impression cast and the subtalar joint neutral position.

For many years, podiatric physicians have been casting orthotic devices with the foot placed in the subtalar joint neutral position based on work by Root et al. Recent research pertaining to the subtalar joint neutral position during the gait cycle is in disagreement with the theory of Root et al and the inverted subtalar joint neutral casting technique. The current research and a historical perspective of casting techniques will be reviewed to help clarify terminology and to decipher the relationship between the rearfoot position during the gait cycle and casting technique.

Improvements in the design of viscoelastic heel orthoses. A clinical study.

A heel orthosis combining the principles of weight redistribution and shock attenuation was dispensed to 30 subjects with heel pain. Relief or cessation of pain occurred in 73% of the cases. The factor most clearly and significantly associated with the effectiveness of the orthosis was a lesser degree of overweight. Within this prescriptive guideline, such a device has the potential of becoming a cost-effective and time-efficient way of treating heel pain of mechanical origin.

Torsional development of the lower extremity. Implications for in-toe and out-toe treatment.

The authors provide a comprehensive review of the nature, origins, and natural history of torsion of the lower extremity. Norms for children and adults are discussed, along with implications for treatment.

Multiple hereditary osteochondromatosis. Two cases with ankle and knee deformities.

Two case histories of multiple hereditary osteochondromatosis are presented. Multiple hereditary exostoses are the most common inherited systemic disorder of bone. The presence of ankle deformity in 50% of the cases and the potential for malignant transformation make this an important disease for the clinician to be familiar with.

Orthotic variants.

When patients present with problems for which existing devices are not adequate, research is stimulated. However, new methods and devices must improve on the older versions and should not result in variation that is less effective than the original versions. Variants less effective than the originals will be discussed with illustrative examples. Orthoses, prostheses, and pressure-reduction techniques for the diabetic foot will be considered.

Orthoses in the treatment of rearfoot problems.

Orthotic management is helpful in the treatment of most orthopedic conditions involving the rearfoot, including plantar fasciitis, Achilles tendon disorders, posterior tibial tendon dysfunction, flatfoot, ankle sprains, and problems associated with diabetes, arthritis, and equinus disorders. A review of the effectiveness of orthoses in the treatment of these conditions is presented here. An in-depth analysis of the orthotic management of plantar fasciitis and a critical review of foot orthoses for the pronated foot are presented. Also discussed are the rationale and effectiveness of the tension night splint in the treatment of plantar fasciitis, orthotic devices for the different stages of posterior tibial tendon dysfunction, and the various categories of orthoses for off-loading the diabetic foot. The modern ankle brace, the effectiveness of prefabricated versus prescription foot orthoses, and recent developments in the ankle-foot orthosis are also reviewed.