Charles Giamberardino

Surfactant Protein A Modulates Induction of Regulatory T Cells via TGF-β

TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue-specific induction of Tregs in the periphery remains unclear. We observed that surfactant protein A (SP-A)–deficient mice have impaired expression of Foxp3 and fewer CD25+Foxp3+ Tregs after ex vivo stimulation and after stimulation with LPS in vivo. The addition of exogenous SP-A completely reversed this phenotype. Although SP-A is known to inhibit T cell proliferation under certain activation conditions, both IL-2 levels as well as active TGF-β levels increase on extended culture with exogenous SP-A, providing a key mechanism for the maintenance and induction of Tregs. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3+ Tregs in responder T cell populations in a TGF-β–dependent manner. In mice treated with LPS in vivo, Tregs increased ∼160% in wild-type mice compared with only a 50% increase in LPS-treated SP-A−/− mice 8 d after exposure. Taken together, these findings support the hypothesis that SP-A affects T cell immune function by the induction of Tregs during activation.

Lung Effector Memory and Activated CD4+ T Cells Display Enhanced Proliferation in Surfactant Protein A-Deficient Mice during Allergen-Mediated Inflammation

Although many studies have shown that pulmonary surfactant protein (SP)-A functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4+ T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an OVA model of allergic hypersensitivity, SP-A−/− mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared with wild-type counterparts. Although both OVA-exposed groups had similar proportions of CD86+ DCs and Foxp3+ T regulatory cells, the SP-A−/− mice had elevated proportions of CD4+ activated and effector memory T cells in their lungs compared with wild-type mice. Ex vivo recall stimulation of CD4+ T cell pools demonstrated that cells from the SP-A−/− OVA mice had the greatest proliferative and IL-4–producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that CD4+ activated and effector memory T cells expanded to the greatest extent in the lungs of SP-A−/− OVA mice. Taken together, our data suggested that SP-A influences the prevalence, types, and functions of CD4+ T cells in the lungs during allergic inflammation and that SP deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.

Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes approximately 625,000 deaths per year from nervous system infections. Here, we leveraged a unique, genetically diverse population of C. neoformans from sub-Saharan Africa, commonly isolated from mopane trees, to determine how selective pressures in the environment coincidentally adapted C. neoformans for human virulence. Genome sequencing and phylogenetic analysis of 387 isolates, representing the global VNI and African VNB lineages, highlighted a deep, nonrecombining split in VNB (herein, VNBI and VNBII). VNBII was enriched for clinical samples relative to VNBI, while phenotypic profiling of 183 isolates demonstrated that VNBI isolates were significantly more resistant to oxidative stress and more heavily melanized than VNBII isolates. Lack of melanization in both lineages was associated with loss-of-function mutations in the BZP4 transcription factor. A genome-wide association study across all VNB isolates revealed sequence differences between clinical and environmental isolates in virulence factors and stress response genes. Inositol transporters and catabolism genes, which process sugars present in plants and the human nervous system, were identified as targets of selection in all three lineages. Further phylogenetic and population genomic analyses revealed extensive loss of genetic diversity in VNBI, suggestive of a history of population bottlenecks, along with unique evolutionary trajectories for mating type loci. These data highlight the complex evolutionary interplay between adaptation to natural environments and opportunistic infections, and that selection on specific pathways may predispose isolates to human virulence.

Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii

ABSTRACT The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease. Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

Surfactant Protein A Integrates Activation Signal Strength To Differentially Modulate T Cell Proliferation

Pulmonary surfactant lipoproteins lower the surface tension at the alveolar–airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A–mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A−/− mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A–dependent effects are mediated by changes in intracellular Ca2+ levels over time, involving extrinsic Ca2+-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease.

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A–deficient mice on a C57BL/6 background (H2b; SP-A−/−) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A–deficient recipient mice that have undergone an allogeneic BMT [SP-A−/−alloBMT] or SP-A–sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A–deficient recipient mice that have undergone a syngeneic BMT or SP-A–sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A−/− alloBMT or SP-A–sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A−/−alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A−/−alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.

Nitric Oxide Mediates Relative Airway Hyporesponsiveness to Lipopolysaccharide in Surfactant Protein A–Deficient Mice

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

Prevalence, healthcare resource utilization and overall burden of fungal meningitis in the United States

Purpose. Previous epidemiological and cost studies of fungal meningitis have largely focused on single pathogens, leading to a poor understanding of the disease in general. We studied the largest and most diverse group of fungal meningitis patients to date, over the longest follow‐up period, to examine the broad impact on resource utilization within the United States. Methodology. The Truven Health Analytics MarketScan database was used to identify patients with a fungal meningitis diagnosis in the United States between 2000 and 2012. Patients with a primary diagnosis of cryptococcal, Coccidioides, Histoplasma, or Candida meningitis were included in the analysis. Data concerning healthcare resource utilization, prevalence and length of stay were collected for up to 5 years following the original diagnosis. Results. Cryptococcal meningitis was the most prevalent type of fungal meningitis (70.1 % of cases over the duration of the study), followed by coccidioidomycosis (16.4 %), histoplasmosis (6.0 %) and candidiasis (7.6 %). Cryptococcal meningitis and candidiasis patients accrued the largest average charges (

Increased Nitric Oxide Production Prevents Airway Hyperresponsiveness in Caveolin-1 Deficient Mice Following Endotoxin Exposure.

103 236 and

Novel Treatment of Cryptococcal Meningitis via Neurapheresis Therapy

103 803, respectively) and spent the most time in the hospital on average (70.6 and 79 days). Coccidioidomycosis and histoplasmosis patients also accrued substantial charges and time in the hospital (