Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα −/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans , whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα −/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα −/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood

Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain.

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans

Early fungal growth control in pulmonary infection with <i>C. neoformans</i> in the presence of IL-4Rα signaling.

<p>Wild-type (WT, open circle) and IL-4Rα^−/− (gray circle) mice on C57BL/6J background were infected intranasally with <i>C. neoformans</i>. Analysis of fungal burdens in the lung was done at different days <i>post infectionem</i> (dpi) as indicated. Shown is data from n = 7 mice per group from one representative of three independent experiments (14 dpi) or from two independent experiments (7; 21 and 42 dpi). Statistical analysis was done using the unpaired Student's t-test (7 dpi) or Mann-Whitney test. *P<0.05; **P<0.01.</p

In the presence of IL-4Rα elevated pulmonary chemokine expression, IFN-γ mRNA expression and NO production.

<p>Following infection, WT (open circle) and IL-4Rα^−/− (gray circle) mice were sacrificed at the time points indicated (A, C) or at 14 days after infection (B). RT-qPCR analyses were done to determine the expression of mRNAs as indicated. Data are derived from one (A, C, 0 and 7 dpi) or one representative out of two (A, 14 dpi) or three (C, 14 dpi) experiments (n = 6–7 mice per genotype and experiment). Statistical analysis was done using the unpaired Student's t-test (ns, not significant; *P<0.05; **P<0.01; ***P<0.001 (A, C)). The concentration of nitric oxide (NO) in cell culture supernatants was determined using the Griess reaction. Pooled data from two different experiments are shown. Dotted line represents detection limit. Statistical analysis was done using the Mann-Whitney test. **P<0.01 (B).</p