TLR Signaling Rescues Fungicidal Activity in Syk-Deficient Neutrophils.

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we used an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with TLR agonist signaling. LPS, a known immunomodulatory molecule derived from Gram-negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrates partial rescue of fungicidal activity, including phagocytosis, degranulation, and neutrophil swarming, but not reactive oxygen species production against C. albicans, in part due to c-Fos activation. Similarly, LPS priming of human neutrophils rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton's tyrosine kinase (Btk), both critical kinases in the innate immune response to fungi. In vivo, neutropenic mice were reconstituted with wild-type or Syk-deficient neutrophils and challenged i.p. with C. albicans. In this model, LPS improved wild-type neutrophil homing to the fungal challenge, although Syk-deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Cytokine Augmentation Reverses Transplant Recipient Neutrophil Dysfunction Against the Human Fungal Pathogen Candida albicans.

BACKGROUND: Solid organ transplant (SOT) and stem cell transplant (SCT) recipients are at increased risk of invasive fungal disease despite normal neutrophil counts. Here, we measure neutrophil anti-Candida activity. METHODS: Twenty-one SOT and 19 SCT recipients were enrolled 2-4 months posttransplant and compared to 23 healthy control patients (HC). Neutrophils were coincubated with Candida albicans, and percentage killing and swarming responses were measured. RESULTS: Neutrophils from transplant patients had decreased fungicidal capacity compared to HC (42%, 43%, and 72% for SCT, SOT, and HC, respectively; SCT vs HC: P < .0001; SOT vs HC: P < .0001; SOT vs SCT: P = .8), including diminished ability to control hyphal growth (HC vs SOT: 0.1455 vs 0.3894, P ≤ .001; HC vs SCT: 0.1455 vs 0.6295, P ≤ .0001, respectively). Serum from SCT, but not SOT, recipients, inhibited the ability of HC neutrophils to control C. albicans (37%, 45%, and 55% for SCT, SOT, and HC, respectively). Neutrophils' control of hyphal growth was partially restored with granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor. CONCLUSIONS: Despite normal circulating numbers, our data suggest that neutrophils from SOT and SCT recipients mount dysfunctional responses against C. albicans. Intrinsic neutrophil changes and extrinsic serum factors may be responsible for the dysfunction, which is partially reversed with cytokine augmentation.

Blue Light Deactivation of Catalase Suppresses Candida Hyphae Development Through Lipogenesis Inhibition.

Hyphae formation is a key step for fungal penetration into epithelial cells and escaping from macrophages or neutrophils. We found that 405 nm light-induced catalase deactivation results in the inhibition of hyphae growth in Candida albicans. The treatment is capable of inhibiting hyphae growth across multiple hyphae-producing Candida species. Metabolic studies on light-treated C. albicans reveal that light treatment results in a strong reduction in both lipid and protein metabolism. A significant decrease in unsaturated and saturated fatty acids was detected through mass spectroscopy, indicating that the suppression of hyphae through light-induced catalase deactivation may occur through inhibition of lipid metabolism. Initial in vivo tests indicate that blue light treatment can suppress the hyphae forming capabilities of C. albicans within murine abrasion infections. Together, these findings open new avenues for the treatment of Candida fungal infections by targeting their dimorphism.

Transfusable neutrophil progenitors as cellular therapy for the prevention of invasive fungal infections.

The use of mature neutrophil (granulocyte) transfusions for the treatment of neutropenic patients with invasive fungal infections (IFIs) has been the focus of multiple clinical trials. Despite these efforts, the transfusion of mature neutrophils has resulted in limited clinical benefit, likely owing to problems of insufficient numbers and the very short lifespan of these donor cells. In this report, we employed a system of conditionally immortalized murine neutrophil progenitors that are capable of continuous expansion, allowing for the generation of unlimited numbers of homogenous granulocyte-macrophage progenitors (GMPs). These GMPs were assayed in vivo to demonstrate their effect on survival in 2 models of IFI: candidemia and pulmonary aspergillosis. Mature neutrophils derived from GMPs executed all cardinal functions of neutrophils. Transfused GMPs homed to the bone marrow and spleen, where they completed normal differentiation to mature neutrophils. These neutrophils were capable of homing and extravasation in response to inflammatory stimuli using a sterile peritoneal challenge model. Furthermore, conditionally immortalized GMP transfusions significantly improved survival in models of candidemia and pulmonary aspergillosis. These data confirm the therapeutic benefit of prophylactic GMP transfusions in the setting of neutropenia and encourage development of progenitor cellular therapies for the management of fungal disease in high-risk patients.

Baseline procalcitonin as a predictor of bacterial infection and clinical outcomes in COVID-19: A case-control study.

PURPOSE: Coronavirus disease-2019 (COVID-19) is associated with a wide spectrum of clinical symptoms including acute respiratory failure. Biomarkers that can predict outcomes in patients with COVID-19 can assist with patient management. The aim of this study is to evaluate whether procalcitonin (PCT) can predict clinical outcome and bacterial superinfection in patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). METHODS: Adult patients diagnosed with SARS-CoV-2 by nasopharyngeal PCR who were admitted to a tertiary care center in Boston, MA with SARS-CoV-2 infection between March 17 and April 30, 2020 with a baseline PCT value were studied. Patients who were presumed positive for SARS-CoV-2, who lacked PCT levels, or who had a positive urinalysis with negative cultures were excluded. Demographics, clinical and laboratory data were extracted from the electronic medical records. RESULTS: 324 patient charts were reviewed and grouped by clinical and microbiologic outcomes by day 28. Baseline PCT levels were significantly higher for patients who were treated for true bacteremia (p = 0.0005) and bacterial pneumonia (p = 0.00077) compared with the non-bacterial infection group. Baseline PCT positively correlated with the NIAID ordinal scale and survival over time. When compared to other inflammatory biomarkers, PCT showed superiority in predicting bacteremia. CONCLUSIONS: Baseline PCT levels are associated with outcome and bacterial superinfection in patients hospitalized with SARS-CoV-2.

Loss of Coordinated Neutrophil Responses to the Human Fungal Pathogen, Candida albicans, in Patients With Cirrhosis.

Neutrophils are the most abundant white blood cell in the body and are key participants in the defense against fungal infections. Fungal infections occur often in patients with cirrhosis and are associated with increased 30-day and 90-day mortality. Previous studies have shown that specific neutrophil functions are abnormal in patients with cirrhosis, although the extent of neutrophil dysfunction is not well understood. We tested the ability of neutrophils from 21 hospitalized patients with cirrhosis and 23 healthy control patients to kill Candida albicans, a common fungal pathogen in patients with cirrhosis. Using an assay, we also measured the ability of neutrophils to coordinate multicellular, synchronized control of C. albicans hyphae through a process known as swarming. We found that neutrophils from patients with cirrhosis have significantly decreased fungicidal capacity compared with healthy control neutrophils (53% vs. 74%, P < 0.0001) and diminished ability to control hyphal growth normalized as a ratio to healthy control (0.22 vs. 0.65, P < 0.0001). Moreover, serum from patients with cirrhosis decreases the ability of healthy control neutrophils to kill C. albicans (from 60% to 41%, P < 0.003). Circulating concentration of the inflammatory cytokines tumor necrosis factor α, interleukin-6, and interleukin-8 were found to be significantly elevated in patients with cirrhosis compared to healthy controls. Following pretreatment with granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor, neutrophil function was restored to almost that of healthy controls. Conclusion: Our data establish profound neutrophil dysfunction against, and altered swarming to, C. albicans in patients with cirrhosis. This dysfunction can be partially reversed with cytokine augmentation ex vivo.

Light- and Melanin Nanoparticle-Induced Cytotoxicity in Metastatic Cancer Cells.

Melanin nanoparticles are known to be biologically benign to human cells for a wide range of concentrations in a high glucose culture nutrition. Here, we show cytotoxic behavior at high nanoparticle and low glucose concentrations, as well as at low nanoparticle concentration under exposure to (nonionizing) visible radiation. To study these effects in detail, we developed highly monodispersed melanin nanoparticles (both uncoated and glucose-coated). In order to study the effect of significant cellular uptake of these nanoparticles, we employed three cancer cell lines: VM-M3, A375 (derived from melanoma), and HeLa, all known to exhibit strong macrophagic character, i.e., strong nanoparticle uptake through phagocytic ingestion. Our main observations are: (i) metastatic VM-M3 cancer cells massively ingest melanin nanoparticles (mNPs); (ii) the observed ingestion is enhanced by coating mNPs with glucose; (iii) after a certain level of mNP ingestion, the metastatic cancer cells studied here are observed to die-glucose coating appears to slow that process; (iv) cells that accumulate mNPs are much more susceptible to killing by laser illumination than cells that do not accumulate mNPs; and (v) non-metastatic VM-NM1 cancer cells also studied in this work do not ingest the mNPs, and remain unaffected after receiving identical optical energy levels and doses. Results of this study could lead to the development of a therapy for control of metastatic stages of cancer.

Candida albicans necrotizing fasciitis following elective surgery.

Necrotizing fasciitis is a potentially fatal soft tissue infection that requires prompt clinical suspicion, pharmacological and surgical interventions. Bacterial pathogens, such as beta-hemolytic streptococcus and Staphylococcus aureus, are the main etiology of necrotizing fasciitis, however, rare cases caused by fungal pathogens, such as Candida albicans, have been reported following trauma. Here, we present the first case of C. albicans necrotizing fasciitis following an elective surgical procedure in an immunocompetent adult.

Spleen Tyrosine Kinase Is a Critical Regulator of Neutrophil Responses to Candida Species.

Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans, Candida glabrata, and Candida auris Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics.IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.

Intent to participate in future cervical cancer screenings is lower when satisfaction with the decision to be vaccinated is neutral.

BACKGROUND: HPV vaccination programs have adversely affected participation in future cervical cancer screening. The purpose of this study is to determine the influence of decision satisfaction with accepting/rejecting the HPV vaccine, as well as traditional clinical factors, on the intent to participate in future screening. METHODS AND FINDINGS: From January 2011 through August 2012 women 18-26 years old presenting for health care in an urban college student health and wellness clinic in the US Midwest were asked to complete a descriptive and medical history survey including a six element decisional satisfaction survey scored on 5-point Likert scales, where the intent to participate in future cervical cancer screening was measured. Of the 568 women who completed the decisional satisfaction survey, 17% of those <21 years and 7% ≥ 21 years indicated no intent to participate in future cervical cancer screenings. Among women of current screening age, the univariate risk factors of race/ethnicity, contraceptive use, number of lifetime sexual partners, and receipt of HPV vaccine were not predictors of intent for future cervical cancer screening. Instead, only a history of a prior Pap test was a significant positive predictor and only a decisional satisfaction of 'neutral' (Likert score = 3) for any of the four decisional satisfaction elements was a significant negative predictor. For the decisional satisfaction element "best for me personally", there was a 78% decreased likelihood of intending to participate in future screening if the satisfaction was neutral rather than firm (aOR = 0.22, 95% CI: 0.05-0.91) and a 26 fold increased likelihood if she had had a prior Pap test (aOR = 26, 95% CI: 5-133). CONCLUSIONS: HPV vaccination implementation programs must help women be the owner of their decision around HPV vaccination and understand the importance of future participation in cervical cancer screening.

Women have a preference for their male partner to be HPV vaccinated.

BACKGROUND: Peer influence and social networking can change female adolescent and young adult behavior. Peer influence on preferences for male human papillomavirus (HPV) vaccination has not been documented. The primary aim of this study was to determine if women had preferences about male sexual partner HPV vaccination receipt. METHODS AND FINDINGS: A prospective survey of women 18-26 years of age was conducted at an urban university student health clinic. Education about the two HPV vaccines, cervical cancer and genital warts was provided. Women self-reported their demographic and medical history data, as well as their own preferences for HPV vaccine and their preferences for their male partner HPV vaccine using a 5 point Likert scale. 601 women, mean age of 21.5 years (SD 2.4), participated between 2011 and 2012. Nearly 95% of respondents were heterosexual; condoms and contraceptives were used in over half of the population. Regardless of the woman's vaccination status, women had significantly higher (strongly agree/agree) preferences for the male partner being vaccinated with HPV4 than not caring if he was vaccinated (63.6% vs. 13.1%, p<0.001). This preference was repeated for sexual risk factors and past reproductive medical history. Women who received HPV4 compared to those choosing HPV2 had a significantly lower proportion of preferences for not caring if the male partner was vaccinated (13% vs. 22%, p = 0.015). CONCLUSIONS: Women preferred a HPV vaccinated male partner. Peer messaging might change the male HPV vaccination uptake.

Quantifying the decisional satisfaction to accept or reject the Human Papillomavirus (HPV) vaccine: a preference for cervical cancer prevention.

OBJECTIVE: Only a portion of the US population is willing to consider HPV vaccination to date. The primary aim of this study is to determine the decisional satisfaction associated with HPV vaccination. STUDY DESIGN: This is a prospective survey conducted at an urban college where women 18-26 years old completed a decisional satisfaction survey about their HPV vaccine experience. RESULTS: Regardless of the decision to accept or reject HPV vaccination, the decisional satisfaction was very high (mean 5-item score = 21.2 (SD 3.8)). Women without HPV vaccination were decisionally neutral significantly more often than those already vaccinated; 22% were decisionally neutral for the option to accept HPV vaccination at that visit. Cervical cancer prevention was preferred significantly more often than genital wart prevention in all analyses. CONCLUSIONS: Targeting those who are decisionally neutral about HPV vaccination may result in a higher uptake of HPV vaccination.