Photoactivation of hypericin generates singlet oxygen in mitochondria and inhibits succinoxidase.

Photosensitized inhibition of mitochondrial succinoxidase by hypericin was measured in vitro and found to be drug-dose, light-dose, and wavelength dependent. Singlet oxygen generation, monitored using the singlet oxygen trap tetramethylethylene, and oxygen consumption in isolated mitochondria sensitized by hypericin were also light-dose and wavelength dependent. Unequivocal evidence for the generation of singlet oxygen was obtained using kinetic isotope ratios of products from the reaction between singlet oxygen and geminally deuterated tetramethylethylene. An action spectrum for the inhibition of succinoxidase was measured at wavelengths between 400 and 700 nm and found to parallel the recorded visible absorption spectrum of hypericin in isolated mitochondria. The greatest singlet oxygen generation, oxygen consumption, and succinoxidase inhibition occurred with white light or 600 nm irradiation. These data are consistent with a type II singlet-oxygen-mediated mechanism for hypericin induced photosensitized inhibition of mitochondrial succinoxidase.

Identification of anti-alpha toxin monoclonal antibodies that reduce the severity of Staphylococcus aureus dermonecrosis and exhibit a correlation between affinity and potency.

Staphylococcus aureus alpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (K(D)) ranging from 0.50 to 15 nM. In an S. aureus dermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC(50)) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate that in vitro potency of anti-AT MAbs predicts in vivo potency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.

Bivalency is required for anticapsular monoclonal antibodies to optimally suppress activation of the alternative complement pathway by the Cryptococcus neoformans capsule.

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.

Capsular reactions of Cryptococcus neoformans with polyspecific and oligospecific polyclonal anticapsular antibodies.

Monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans produce distinct capsular reactions and have biological activities that are determined by serotype specificity. In the present study, polyclonal rabbit anticapsular antibodies were cross-absorbed to produce serotype specificities similar to those of monoclonal antibodies. The results showed that polyclonal and monoclonal antibodies with similar serotype specificities have similar capsular reactions and biological activities.

An insect model for assessing mercury toxicity: effect of mercury on antioxidant enzyme activities of the housefly (Musca domestica) and the cabbage looper moth (Trichoplusia ni).

The effect of mercury as Hg2Cl2 and HgCl2 on the antioxidant enzyme levels and its toxicity was investigated in an insect model comprised of adult females of the common housefly, Musca domestica, and fourth-instar larvae of the cabbage looper moth, Trichoplusia ni. HgCl2 was found to be more toxic than Hg2Cl2 to both M. domestica and T. ni. The LC50s for M. domestica were 1.17% and 0.38% w/v concentration for Hg2Cl2 and HgCl2, respectively. For the more tolerant T. ni, the LC50S were 5.15% for Hg2Cl2 and 0.96% w/w concentration for HgCl2. The minimally acute LC5 dose of both oxidation states of Hg was approximately 0.005% for both insects (w/v for M. domestica and w/w for T. ni). At the LC5, both forms of Hg significantly induced the activity of superoxide dismutase in both insect species. Catalase was induced by both Hg2Cl2 and HgCl2 in M. domestica but was only induced by HgCl2 in T. ni. Glutathione-S-transferase, its peroxidase activity, and glutathione reductase activities were also significantly altered in most cases by Hg in both insects although the pattern of alternation was different between the two insects. It is evident that mercury induces oxidative stress in insects as it does in vertebrates. Our findings suggest that insects may serve as a valuable, non-mammalian model species to assess Hg-induced oxidative stress as a component of environmental toxicity.

Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans.

The capsular swelling or quellung reaction was reported almost 100 years ago and described the effect of Abs on the appearance of microbial capsules. Despite widespread use to assess Ab binding to capsules, relatively little is known as to the mechanism of this effect or its biological consequences. The fungus Cryptococcus neoformans is an attractive system to study capsule reactions because it has a large polysaccharide capsule that is readily visible by light microscopy. When viewed by differential interference contrast microscopy, binding of mAb to C. neoformans cells produced two distinct capsular reactions that depended on the Ab epitope specificity and the yeast serotype. In the first pattern, termed "rim," the capsule appears transparent with a highly refractive outer edge. In the second pattern, termed "puffy," the capsule appears opaque and lacks a highly refractive outer rim. mAbs that bind with a rim pattern suppress the overall rate of C3 deposition on the yeast via the classical and alternative complement pathways. In contrast, mAbs that bind with a puffy pattern do not affect C3 deposition. Protective and nonprotective IgM mAbs produce rim and puffy patterns, respectively. These results indicate that: 1) capsule reactions are a consequence of Ab-induced changes in capsular refractive index; 2) the type of capsule reaction depends on the Ab specificity; and 3) Ab-induced changes in refractive index correlate with biological activities important for host defense against C. neoformans. Our results provide the first evidence associating distinct capsule reaction patterns with Ab biological activity.

Dichlone-induced oxidative stress in a model insect species, Spodoptera eridania.

Southern armyworm, Spodoptera eridania, larvae were provided ad libitum 0.002-0.25% w/w dichlone, 2,3-dichloro-1,4-naphthoquinone (CNQ). Larval mortality occurred in a time-and-dose dependent manner, with an LC17 of 0.01% and an LC50 of 0.26% CNQ at day-5. Extracts of larvae fed control, 0.01, and 0.25% CNQ diets for 5 days were assayed for antioxidant enzymes. While 0.01% CNQ had a mild effect, 0.25% CNQ profoundly increased levels of all antioxidant enzymes that were examined. The increases as compared to control were: 5.3-, 1.9-, 3.2-, 2.6-, 2.8-, and 3.5-fold higher for superoxide dismutase, catalase, glutathione transferase and its peroxidase activity, glutathione reductase and DT-diaphorase, respectively. At 0.01% CNQ, the thiobarbituric acid reactive substances (TBARS) were similar to the control group. However, despite the induction from 0.25% CNQ of all enzymes examined, the lipid peroxidation was not attenuated; the TBARS were 29.7% over the control value. High mortalities and CNQ-induced pathologies reflected in retarded growth, wasting syndrome, and diuresis clearly indicated that the insect sustained severe oxidant-induced injuries before appropriate defenses were fully mobilized. Thus, this quinone causes an oxidative stress in a model insect species analogous to that observed in mammalian species.

An insect model for assessing oxidative stress related to arsenic toxicity.

The potential usefulness of an insect model to evaluate oxidative stress induced by environmental pollutants was examined with trivalent arsenic (As3+, NaAsO2) and pentavalent arsenic (As5+, Na2HAsO4) in adult female house flies, Musca domestica, and fourth-instar cabbage loopers, Trichoplusia ni. M. domestica was highly susceptible to both forms of arsenic following 48 h exposure in the drinking water with LC50s of 0.008 and 0.011% w/v for As3+ and As5+, respectively. T. ni larvae were susceptible to dietary As3+ with an LC50 of 0.032% w/w but seem to tolerate As5+ well with an LC50 of 0.794% concentration after 48 h exposure. The minimally acute LC5 dose of both As3+ and As5+ varied considerably but averaged 0.005% for both insects. The potential of both valencies of arsenic for inducing oxidative stress in the insects exposed ad libitum to approximately LC5 levels was assessed. The parameters examined were the alterations of the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), the peroxidase activity of glutathione transferase (GSTPX), and glutathione reductase (GR), and increases in lipid peroxidation and protein oxidation. SOD (1.3-fold), GST (1.6-fold), and GR (1.5-fold) were induced by As3+ in M. domestica but CAT and GSTPX were not affected. As5+ had no effect on M. domestica. In T. ni, the antioxidant enzyme activities were not affected by As3+ except for SOD which was suppressed by 29.4% and GST which was induced by 1.4-fold. As5+ had no effect except the suppression of SOD by 41.2%. Lipid peroxidation and protein oxidation, which represent stronger indices of oxidative stress, were elevated in both insects by up to 2.9-fold. However, based on the antioxidant enzyme response to the arsenic anions, the mode of action of arsenic induced oxidative stress may differ between the two insects. Until this aspect is further clarified, evidence at this time favors the prospect of As3+ as a pro-oxidant, especially for M. domestica.

Mitomycin C induced alterations in antioxidant enzyme levels in a model insect species, Spodoptera eridania.

1. An insect species, the southern armyworm Spodoptera eridania, was used as an in vivo model to examine mitomycin C's (MMC) pro-oxidant effect reflected in alterations of antioxidant enzymes. 2. Following a 2-day exposure to 0.01 and 0.05% w/w dietary concentrations, MMC only induced superoxide dismutase activity. All other enzyme activities were not affected, indicating oxidative stress was mild. 3. Following a 5-day exposure to 0.05% w/w dietary MMC, the activities of superoxide dismutase, glutathione-S-transferase and its peroxidase activity and DT-diaphorase were induced. GR activity was not altered. The high constitutive catalase activity was also not affected. These responses of S. eridania's antioxidant enzymes are analogous to those of mammalian systems in alleviating MMC-induced oxidative stress. 4. S. eridania emerges as an appropriate non-mammalian model for initial and cost-effective screening of drug-induced oxidative stress.

Activation of the alternative complement pathway by fungal melanins.

Melanins are complex biological pigments formed by the oxidative polymerization of phenolic and/or indolic compounds. These pigments have been implicated in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases. Recent studies have demonstrated that melanins have antigenic and anti-inflammatory properties. These findings led us to further explore the interaction of melanins with the immune system. Melanin particles ("ghosts") were isolated from in vitro-melanized Cryptococcus neoformans cells and Aspergillus niger conidia and then incubated in normal human serum containing (125)I-labeled complement C3. The results demonstrated deposition of C3 fragments onto the melanin ghosts as early as 1 min after incubation, with maximum deposition occurring after 12 min for C. neoformans-derived melanin ghosts and after 25 min for A. niger-derived melanin ghosts. The blocking of classical pathway activation did not affect the kinetics or total deposition of C3 onto the melanin ghosts, indicating that melanins activate complement through the alternative pathway. Immunofluorescence analysis of lungs from BALB/c mice injected intratracheally with C. neoformans-derived melanin ghosts demonstrated deposition of C3 fragments onto the ghosts. Small granulomas were also observed surrounding the ghosts. However, melanization of the C. neoformans cell wall did not alter the kinetics or total deposition of C3 fragments onto the fungal cells. The finding that melanin surfaces can activate the complement system suggests a potential mechanism for the pathogenesis of some degenerative and/or autoimmune processes that involve melanized cells as well as another potential role for melanin in the virulence of melanin-producing microorganisms.

Characterization of anticapsular monoclonal antibodies that regulate activation of the complement system by the Cryptococcus neoformans capsule.

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors.

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.

Complement is essential for protection by an IgM and an IgG3 monoclonal antibody against experimental, hematogenously disseminated candidiasis.

The incidence of life-threatening, hematogenously disseminated candidiasis, which is predominantly caused by Candida albicans, parallels the use of modern medical procedures that adversely affect the immune system. Limited antifungal drug choices and emergence of drug-resistant C. albicans strains indicate the need for novel prevention and therapeutic strategies. We are developing vaccines and Abs that enhance resistance against experimental candidiasis. However, the prevalence of serum anti-Candida Abs in candidiasis patients has led to the misconception that Abs are not protective. To explain the apparent discrepancy between such clinical observations and our work, we compared functional activities of C. albicans-specific protective and nonprotective mAbs. Both kinds of Abs are agglutinins that fix complement and are specific for cell surface mannan, but the protective Abs recognize beta-mannan, and the nonprotective Ab is specific for alpha-mannan. By several indirect and direct measures, the protective mAbs more efficiently bind complement factor C3 to the yeast cell than do nonprotective Ab. We hypothesize that the C3 deposition causes preferential association of blood-borne fungi with host phagocytic cells that are capable of killing the fungus. We conclude from these results that the protective potential of Abs is dependent on epitope specificity, serum titer, and ability to rapidly and efficiently fix complement to the fungal surface. The mechanism of protection appears to be associated with enhanced phagocytosis and killing of the fungus.