Introduction

Aureobasidium pullulans is a saprophytic fungus that is widely distributed in the environment, and in the right host can be an opportunistic human pathogen.

Presentation of case

A 66-year-old man with Crohn's disease with a single kidney, and requiring total parenteral nutrition via a Hickman catheter, was admitted with a 10-week history of progressive shortness of breath, fevers and weight loss. Chest imaging demonstrated new multifocal lung parenchymal opacities compatible with septic pulmonary emboli. Blood culture grew a yeast-like organism that transformed into a black mold on subculture, eventually identified as A. pullulans. Due to triazole resistance, the patient was treated with liposomal amphotericin and micafungin. Serum (1,3)-β-d-glucan level was used to monitor therapy, initially measured at >500 pg/mL and decreasing to 66 pg/mL after one year of therapy.

Discussion

We describe the successful treatment of a case of catheter related fungemia and septic pulmonary emboli due A. pullulans. While initially appearing as an oval yeast on blood culture, subsequent growth as a black mold led to identification of the fungus as A. pullulans. The infection was cured with a combination of antifungal agents, even though the foreign body could not be safely removed. Nephrotoxicity required dosing adjustment of the amphotericin to biweekly during the maintenance phase of treatment. The serum (1,3)-β-d-glucan level proved to be useful in monitoring response to therapy.

Conclusion

We report here successful treatment of a disseminated A. pullulans infection with an induction and maintenance approach to liposomal amphotericin dosing, and monitoring response to therapy with serum (1,3)-β-d-glucan levels.

Objective

Lack of judicious testing can result in the incorrect diagnosis of Clostridioides difficile infection (CDI), unnecessary CDI treatment, increased costs and falsely augmented hospital-acquired infection (HAI) rates. We evaluated facility-wide interventions used at the VA San Diego Healthcare System (VASDHS) to reduce healthcare-onset, healthcare-facility-associated CDI (HO-HCFA CDI), including the use of diagnostic stewardship with test ordering criteria.

Design

We conducted a retrospective study to assess the effectiveness of measures implemented to reduce the rate of HO-HCFA CDI at the VASDHS from fiscal year (FY)2015 to FY2018.

Interventions

Measures executed in a stepwise fashion included a hand hygiene initiative, prompt isolation of CDI patients, enhanced terminal room cleaning, reduction of fluoroquinolone and proton-pump inhibitor use, laboratory rejection of solid stool samples, and lastly diagnostic stewardship with C. difficile toxin B gene nucleic acid amplification testing (NAAT) criteria instituted in FY2018.

Results

From FY2015 to FY2018, 127 cases of HO-HCFA CDI were identified. All rate-reducing initiatives resulted in decreased HO-HCFA cases (from 44 to 13; P ≤ .05). However, the number of HO-HCFA cases (34 to 13; P ≤ .05), potential false-positive testing associated with colonization and laxative use (from 11 to 4), hospital days (from 596 to 332), CDI-related hospitalization costs (from $2,780,681 to $1,534,190) and treatment cost (from $7,158 vs $1,476) decreased substantially following the introduction of diagnostic stewardship with test criteria from FY2017 to FY2018.

Conclusions

Initiatives to decrease risk for CDI and diagnostic stewardship of C. difficile stool NAAT significantly reduced HO-HCFA CDI rates, detection of potential false-positives associated with laxative use, and lowered healthcare costs. Diagnostic stewardship itself had the most dramatic impact on outcomes observed and served as an effective tool in reducing HO-HCFA CDI rates.

Encephalopathy complicates beta-lactam therapy, particularly with impaired renal function, though no studies have reported ceftaroline-associated encephalopathy. Among 28 patients with estimated glomerular filtration rates <30 mL/min who received ≥5 days of ceftaroline, 3 developed encephalopathy. Ceftaroline, when dosed supra-therapeutically for serious infections, may be a cause of antibiotic-associated encephalopathy.

While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I - T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.

In cancer, proteoglycans have been found to play roles in facilitating the actions of growth factors, and effecting matrix invasion and remodeling. However, little is known regarding the genetic and functional importance of glycan chains displayed by proteoglycans on dendritic cells (DCs) in cancer immunity. In lung carcinoma, among other solid tumors, tumor-associated DCs play largely subversive/suppressive roles, promoting tumor growth and progression. Herein, we show that targeting of DC glycan sulfation through mutation in the heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1) in mice increased DC maturation and inhibited trafficking of DCs to draining lymph nodes. Lymphatic-driven DC migration and chemokine (CCL21)-dependent activation of a major signaling pathway required for DC migration (as measured by phospho-Akt) were sensitive to Ndst1 mutation in DCs. Lewis lung carcinoma tumors in mice deficient in Ndst1 were reduced in size. Purified CD11c+ cells from the tumors, which contain the tumor-infiltrating DC population, showed a similar phenotype in mutant cells. These features were replicated in mice deficient in syndecan-4, the major heparan sulfate proteoglycan expressed on the DC surface: Tumors were growth-impaired in syndecan-4-deficient mice and were characterized by increased infiltration by mature DCs. Tumors on the mutant background also showed greater infiltration by NK cells and NKT cells. These findings indicate the genetic importance of DC heparan sulfate proteoglycans in tumor growth and may guide therapeutic development of novel strategies to target syndecan-4 and heparan sulfate in cancer.

Abstract Background Lymph node metastasis constitutes a key event in tumor progression. The molecular control of this process is poorly understood. Heparan sulfate is a linear polysaccharide consisting of unique sulfate-modified disaccharide repeats that allow the glycan to bind a variety of proteins, including chemokines. While some chemokines may drive lymphatic trafficking of tumor cells, the functional and genetic importance of heparan sulfate as a possible mediator of chemokine actions in lymphatic metastasis has not been reported. Results We applied a loss-of-function genetic approach employing lymphatic endothelial conditional mutations in heparan sulfate biosynthesis to study the effects on tumor-lymphatic trafficking and lymph node metastasis. Lymphatic endothelial deficiency in N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in sulfating nascent heparan sulfate chains, resulted in altered lymph node metastasis in tumor-bearing gene targeted mice. This occurred in mice harboring either a pan-endothelial Ndst1 mutation or an inducible lymphatic-endothelial specific mutation in Ndst1. In addition to a marked reduction in tumor metastases to the regional lymph nodes in mutant mice, specific immuno-localization of CCL21, a heparin-binding chemokine known to regulate leukocyte and possibly tumor-cell traffic, showed a marked reduction in its ability to associate with tumor cells in mutant lymph nodes. In vitro modified chemotaxis studies targeting heparan sulfate biosynthesis in lymphatic endothelial cells revealed that heparan sulfate secreted by lymphatic endothelium is required for CCL21-dependent directional migration of murine as well as human lung carcinoma cells toward the targeted lymphatic endothelium. Lymphatic heparan sulfate was also required for binding of CCL21 to its receptor CCR7 on tumor cells as well as the activation of migration signaling pathways in tumor cells exposed to lymphatic conditioned medium. Finally, lymphatic cell-surface heparan sulfate facilitated receptor-dependent binding and concentration of CCL21 on the lymphatic endothelium, thereby serving as a mechanism to generate lymphatic chemokine gradients. Conclusions This work demonstrates the genetic importance of host lymphatic heparan sulfate in mediating chemokine dependent tumor-cell traffic in the lymphatic microenvironment. The impact on chemokine dependent lymphatic metastasis may guide novel therapeutic strategies.

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane "rippling" along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.

TNF-stimulated gene-6 (TSG-6) is a multifunctional protein secreted in response to pro-inflammatory stimuli by a wide range of cells, including neutrophils, monocytes, and endothelial cells. It has been shown to mediate anti-inflammatory and protective effects when administered in disease models, in part, by reducing neutrophil infiltration. Human TSG-6 inhibits neutrophil migration by binding CXCL8 through its Link module (Link_TSG6) and interfering with the presentation of CXCL8 on cell-surface glycosaminoglycans (GAGs), an interaction that is vital for the function of many chemokines. TSG-6 was also found to interact with chemokines CXCL11 and CCL5, suggesting the possibility that it may function as a broad specificity chemokine-binding protein, functionally similar to those encoded by viruses. This study was therefore undertaken to explore the ability of TSG-6 to regulate the function of other chemokines. Herein, we demonstrate that Link_TSG6 binds chemokines from both the CXC and CC families, including CXCL4, CXCL12, CCL2, CCL5, CCL7, CCL19, CCL21, and CCL27. We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1-85 nm) broadly correlate with chemokine-GAG affinities. Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs. Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo.

Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL-33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL-33 receptor gene deleted (ST2^-/- ) and MYD88 gene deleted (MYD88^-/- ) mice by exposure to 10% O₂ and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL-33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p < 0.0001) and this SUHX response was attenuated in ST2^-/- mice (29% male p = 0.003; 30% female p = 0.001) and absent in MYD88^-/- mice. Wall thickness was increased in SUHX C57Bl/6J mice (p = 0.005), but not in ST2^-/- or MYD88^-/- mice. Proliferating cells were detected in C57Bl/6J mice by flow cytometry (CD31⁺ /BrDU⁺ ; p = 0.02) and immunofluorescence methods (Ki-67+). IL-33 was increased by SUHX (p = 0.03) but a genotype effect was not observed (p = 0.76). We observed that in hPAECs, IL-33 expression is regulated by both IL-33 and DLL4. These data suggest IL-33/ST2 signaling is essential for the endothelial cell proliferative response in PH.

Rationale

Lymphatic vessel growth is mediated by major prolymphangiogenic factors, such as vascular endothelial growth factor (VEGF-C) and VEGF-D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell membranes and matrix, playing roles in angiogenesis, although little is known about any function(s) in lymphatic remodeling in vivo.

Objective

To explore the genetic basis and mechanisms, whereby heparan sulfate proteoglycans mediate pathological lymphatic remodeling.

Methods and results

Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathological models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling and reduced VEGF-C-mediated protection from starvation-induced apoptosis. Lymphatic endothelial-specific Ndst1 deficiency (in Ndst1(f/f)Prox1(+/CreERT2) mice) was sufficient to inhibit VEGF-C-dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGF receptor-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathological lymphangiogenesis was impaired in Sdc4((-/-)) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGF receptor-3, which was sensitive to glycan disruption. Moreover, VEGF receptor-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency.

Conclusions

These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathological lymphatic remodeling. This may introduce novel future strategies to alter pathological lymphatic-vascular remodeling.