Identification of Irpex and Rhodotorula on surveillance bronchoscopy in a pediatric lung transplant recipient: A case report and review of literature of these atypical fungal organisms.

BACKGROUND: Invasive fungal disease (IFD) is a frequent complication in pediatric lung transplant recipients, occurring in up to 12% of patients in the first year. Risk factors for infection include impaired lung defenses and intense immunosuppressive regimens. While most IFD occurs from Aspergillus, other fungal conidia are continuously inhaled, and infections with fungi on a spectrum of human pathogenicity can occur. CASE REPORT: We report a case of a 17-year-old lung transplant recipient in whom Irpex lacteus and Rhodotorula species were identified during surveillance bronchoscopy. She was asymptomatic and deemed to be colonized by Irpex lacteus and Rhodotorula species following transplant. 2 years after transplantation, she developed a fever, respiratory symptoms, abnormal lung imaging, and histological evidence of acute and chronic bronchitis on transbronchial biopsy. After developing symptoms concerning for a pulmonary infection and graft dysfunction, she was treated for a presumed IFD. Unfortunately, further diagnostic testing could not be performed at this time given her tenuous clinical status. Despite the initiation of antifungal therapy, her graft function continued to decline resulting in a second lung transplantation. CONCLUSIONS: This case raises the concern for IFD in lung transplant recipients from Irpex species. Further investigation is needed to understand the pathogenicity of this organism, reduce the incidence and mortality of IFD in lung transplant recipients, and refine the approach to diagnosis and manage the colonization and isolation of rare, atypical fungal pathogens in immunocompromised hosts.

Smoking induces increased apoptosis in osteoblasts: changes in bone matrix organic components.

Clinical studies demonstrate the impact of smoking on bone tissue fragility and higher incidence of fractures. However, it is not totally understood which physiological mechanisms could be involved in these events. Previously, we showed important changes in bone tissue components in experimental model of cigarette smoke (CS) exposure. CS exposure induces worsening in bone mineralization and a decrease in collagen type I deposition, leading to bone fragility. Considering that the majority of clinical studies described bone structural changes by radiographic images, in this study we performed analyses "in situ" using tissue samples from smokers, former smokers and non-smokers to better understand how the increase in inflammatory mediators induced by smoking exposure could interfere in bone cells activity leading bone structural changes. We observed increased levels of IL-1ß, IL-6 and TNF-α in bone tissue homogenates with a concomitant increase in osteoblast apoptosis in smokers and former smokers compared with non-smokers. Histological changes in both smokers and former smokers were characterized by reduction in collagen type I. Only in smokers, it was observed decrease in trabecular area, suggesting increased bone resorption and increase in collagen type V. These results showed that osteoblasts apoptosis in association with increased bone resorption leads bone structural changes in smokers.

Inflammasome-mediated GSDMD activation facilitates escape of Candida albicans from macrophages.

Candida albicans is the most common cause of fungal sepsis. Inhibition of inflammasome activity confers resistance to polymicrobial and LPS-induced sepsis; however, inflammasome signaling appears to protect against C. albicans infection, so inflammasome inhibitors are not clinically useful for candidiasis. Here we show disruption of GSDMD, a known inflammasome target and key pyroptotic cell death mediator, paradoxically alleviates candidiasis, improving outcomes and survival of Candida-infected mice. Mechanistically, C. albicans hijacked the canonical inflammasome-GSDMD axis-mediated pyroptosis to promote their escape from macrophages, deploying hyphae and candidalysin, a pore-forming toxin expressed by hyphae. GSDMD inhibition alleviated candidiasis by preventing C. albicans escape from macrophages while maintaining inflammasome-dependent but GSDMD-independent IL-1ß production for anti-fungal host defenses. This study demonstrates key functions for GSDMD in Candida's escape from host immunity in vitro and in vivo and suggests that GSDMD may be a potential therapeutic target in C. albicans-induced sepsis.

Perceived barriers to Chagas disease screening among a diverse group of prenatal care providers.

BACKGROUND: Chagas disease is a vector borne infection of poverty endemic to Latin America which affects an estimated 40,000 women of child-bearing age in the United States (US). In the US Chagas disease is concentrated among individuals who have lived in endemic areas. Prenatal diagnosis and treatment are needed to prevent congenital transmission. The objective of this study was to assess perceived barriers to Chagas disease screening among prenatal care providers in Obstetrics/Gynecology and Family Medicine Departments of a tertiary care safety-net hospital caring for a significant at-risk population. METHODOLOGY/PRINCIPAL FINDINGS: An anonymous survey was distributed to 178 Obstetrics/Gynecology and Family Medicine practitioners. Of the 66 respondents, 39% thought Chagas screening was very important, and 48% somewhat important as a public health initiative. One third judged screening patients during clinic visits as very important. Most respondents (64%) reported being familiar with Chagas disease. However, only 32% knew how to order a test and only 22% reported knowing what to do if a test was positive. CONCLUSIONS/SIGNIFICANCE: These findings will be incorporated into measures to facilitate full implementation of Chagas screening, and can inform initiatives at other centers who wish to address this deeply neglected infection among their patient families. Greater integration of information on Chagas disease screening and treatment in medical and nursing education curricula can contribute to addressing this disease with the focus that its potentially fatal sequelae merit.

Th17/Treg imbalance in COPD development: suppressors of cytokine signaling and signal transducers and activators of transcription proteins.

Th17/Treg imbalance contributes to chronic obstructive pulmonary disease (COPD) development and progression. However, intracellular signaling by suppressor of cytokine signaling (SOCS) 1 and SOCS3 and the proteins signal transducer and activator of transcription (STAT) 3 and STAT5 that orchestrate these imbalances are currently poorly understood. Thus, these proteins were investigated in C57BL/6 mice after exposure to cigarette smoke (CS) for 3 and 6 months. The expression of interleukin was measured by ELISA and the density of positive cells in peribronchovascular areas was quantified by immunohistochemistry. We showed that exposure to CS in the 3rd month first induced decreases in the numbers of STAT5+ and pSTAT5+ cells and the expression levels of TGF-ß and IL-10. The increases in the numbers of STAT3+ and pSTAT3+ cells and IL-17 expression occurred later (6th month). These findings corroborate the increases in the number of SOCS1+ cells in both the 3rd and 6th months, with concomitant decreases in SOCS3+ cells at the same time points. Our results demonstrated that beginning with the initiation of COPD development, there was a downregulation of the anti-inflammatory response mediated by SOCS and STAT proteins. These results highlight the importance of intracellular signaling in Th17/Treg imbalance and the identification of possible targets for future therapeutic approaches.

Chronic exposure to diesel particles worsened emphysema and increased M2-like phenotype macrophages in a PPE-induced model.

Chronic exposure to ambient levels of air pollution induces respiratory illness exacerbation by increasing inflammatory responses and apoptotic cells in pulmonary tissues. The ineffective phagocytosis of these apoptotic cells (efferocytosis) by macrophages has been considered an important factor in these pathological mechanisms. Depending on microenvironmental stimuli, macrophages can assume different phenotypes with different functional actions. M1 macrophages are recognized by their proinflammatory activity, whereas M2 macrophages play pivotal roles in responding to microorganisms and in efferocytosis to avoid the progression of inflammatory conditions. To verify how exposure to air pollutants interferes with macrophage polarization in emphysema development, we evaluated the different macrophage phenotypes in a PPE- induced model with the exposure to diesel exhaust particles. C57BL/6 mice received intranasal instillation of porcine pancreatic elastase (PPE) to induce emphysema, and the control groups received saline. Both groups were exposed to diesel exhaust particles or filtered air for 60 days according to the groups. We observed that both the diesel and PPE groups had an increase in alveolar enlargement, collagen and elastic fibers in the parenchyma and the number of macrophages, lymphocytes and epithelial cells in BAL, and these responses were exacerbated in animals that received PPE instillation prior to exposure to diesel exhaust particles. The same response pattern was found inCaspase-3 positive cell analysis, attesting to an increase in cell apoptosis, which is in agreement with the increase in M2 phenotype markers, measured by RT-PCR and flow cytometry analysis. We did not verify differences among the groups for the M1 phenotype. In conclusion, our results showed that both chronic exposure to diesel exhaust particles and PPE instillation induced inflammatory conditions, cell apoptosis and emphysema development, as well as an increase in M2 phenotype macrophages, and the combination of these two factors exacerbated these responses. The predominance of the M2-like phenotype likely occurred due to the increased demand for efferocytosis. However, M2 macrophage activity was ineffective, resulting in emphysema development and worsening of symptoms.

Microenvironmental stimuli induce different macrophage polarizations in experimental models of emphysema.

Macrophages play a pivotal role in the development of emphysema and depending on the microenvironment stimuli can be polarized into M1- or M2-like macrophage phenotypes. We compared macrophage polarizations in cigarette smoke (CS)- and porcine pancreatic elastase (PPE)-induced emphysema models. C57BL/6 mice were subdivided into four experimental groups. In the PPE group, animals received an intranasal instillation of PPE (0.677 IU); in the saline group, animals received an intranasal instillation of saline (0.9%). Animals from both groups were euthanized on day 28. In the CS group, animals were exposed to CS for 30 min, twice a day, 5 days per week for 12 weeks. In the control group, animals received filtered air. We observed an increase in total macrophages for both experimental models. For M1-like macrophage markers, we observed an increase in TNF-α+ and IFN-γ+ cells, Cxcl-9 and Cxcl-10 expressions in PPE and CS groups. Only in the CS group, we detected an increased expression of IL-12b For M2-like macrophages markers we observed a down regulation in IL-10, IL-4, IL-13, Arg1 and Fizz1 and an increase of TGF-ß+ cells in the PPE group, while for the CS group there was an increase in TGF-ß+ cells and IL-10 expression. All exposure groups were compared to their respective controls. In summary, we demonstrated that CS- and PPE-induced models resulted in different microenvironmental stimuli. CS exposure induced an environmental stimulus related to M1- and M2-like macrophage phenotypes similar to previous results described in COPD patients, whereas the elastase-induced model provided an environmental stimulus related only to the M1 phenotype.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

Essential role for vacuolar acidification in Candida albicans virulence.

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V0 subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn(2+) toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.

Conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells by ectopic expression of scleraxis.

Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research.

Bupivacaine, ropivacaine, and morphine: comparison of toxicity on human hamstring-derived stem/progenitor cells.

PURPOSE: Bupivacaine, ropivacaine, and morphine are commonly administered intraarticularly after anterior cruciate ligament (ACL) reconstruction. However, their effects on human tendon stem/progenitor cells (TSPC) have not been studied. Therefore, this study investigates the cytotoxicity of these analgetics on TSPC. METHODS: Cells were isolated from human hamstring grafts of 3 female (age 15, 16 and 59) and 2 male patients (age 16 and 47). Cells were incubated using 0.5% bupivacaine, 0.5/0.75% ropivacaine, and 0.025% morphine. Cell viability was assessed after 0.5, 2, and 6 h using live/dead assay. Metabolic activity and apoptosis were measured by WST- and Annexin-V-FACS-assay after 2 h. RESULTS: Cell viability remained unchanged after 0.5 h in all groups, while treatment with bupivacaine and 0.5/0.75% ropivacaine resulted in a complete cell loss after 6 h. Contrarily, morphine showed no cytotoxic effect. Cell viability and metabolism were significantly reduced after treatment with bupivacaine (22.1; 8.3%) and 0.75% ropivacaine (56.5; 23.8%), while 0.5% ropivacaine and morphine showed no significant difference compared with controls. Apoptosis was significantly induced after incubation with bupivacaine (58.1%) and 0.75% ropivacaine (26.2%), whereas 0.5% ropivacaine only led to a slight induction compared with morphine and controls. CONCLUSIONS: Clinically administered concentrations of bupivacaine (0.5%) and ropivacaine (0.75%) have a significant cytotoxic effect on human TSPC in vitro, while ropivacaine in a concentration of 0.5% has a mild but not significant effect on apoptosis and cell metabolism. In contrast, morphine does not affect cell survival, metabolism, or apoptosis. Knowing that morphine provides comparable to even prolonged pain reduction after ACL reconstruction, the presented in vitro study suggests morphine as a potentially less toxic analgetic drug for intraarticular application in clinical practice.

Mucosal damage and neutropenia are required for Candida albicans dissemination.

Candida albicans fungemia in cancer patients is thought to develop from initial gastrointestinal (GI) colonization with subsequent translocation into the bloodstream after administration of chemotherapy. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but we have hypothesized that both neutropenia and GI mucosal damage are critical for allowing widespread invasive C. albicans disease. We investigated these parameters in a mouse model of C. albicans GI colonization that led to systemic spread after administration of immunosuppression and mucosal damage. After depleting resident GI intestinal flora with antibiotic treatment and achieving stable GI colonization levels of C. albicans, it was determined that systemic chemotherapy with cyclophosphamide led to 100% mortality, whereas selective neutrophil depletion, macrophage depletion, lymphopenia or GI mucosal disruption alone resulted in no mortality. Selective neutrophil depletion combined with GI mucosal disruption led to disseminated fungal infection and 100% mortality ensued. GI translocation and dissemination by C. albicans was also dependent on the organism's ability to transform from the yeast to the hyphal form. This mouse model of GI colonization and fungemia is useful for studying factors of innate host immunity needed to prevent invasive C. albicans disease as well as identifying virulence factors that are necessary for fungal GI colonization and dissemination. The model may also prove valuable for evaluating therapies to control C. albicans infections.

Lung epithelial cells and extracellular matrix components induce expression of Pneumocystis carinii STE20, a gene complementing the mating and pseudohyphal growth defects of STE20 mutant yeast.

Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 (PCSTE20), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Delta mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.

Deficient natural killer cell cytotoxicity in patients with IKK-gamma/NEMO mutations.

NF-kappaB essential modifier (NEMO), also known as IKK-gamma, is a member of the I-kappaB kinase complex responsible for phosphorylating I-kappaB, allowing the release and activation of NF-kappaB. Boys with an expressed NEMO mutation have an X-linked syndrome characterized by hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID). The immunophenotype resulting from NEMO mutation is highly variable, with deficits in both T and B cell responses. We evaluated three patients with NEMO mutations (L153R, Q403X, and C417R) and HED-ID who had evidence of defective CD40 signaling. All three patients had normal percentages of peripheral blood NK cells, but impaired NK cell cytotoxic activity. This was not due to a generalized defect in cytotoxicity because antibody-dependent cellular cytotoxicity was intact. This abnormality was partially reversed by in vitro addition of IL-2, which was also able to induce NF-kappaB activation. In one patient with recurrent cytomegalovirus infections, administration of IL-2 partially corrected the NK cell killing deficit. These data suggest that NEMO participates in signaling pathways leading to NK cell cytotoxicity and that IL-2 can activate NF-kappaB and partially overcome the NK cell defect in patients with NEMO mutations.