Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Modification of Hinge/Transmembrane and Signal Transduction Domains Improves the Expression and Signaling Threshold of GXMR-CAR Specific to Cryptococcus spp.

Chimeric antigen receptors (CARs) redirect T cells to recognize a specific target. CAR components play a pivotal role in antigen specificity, structure stability, expression on cell surface, and induction of cellular activation, which together determine the success of CAR T-cell therapy. CAR products targeting B-cell lymphoma encouraged the development of new CAR applications beyond cancer. For example, our group developed a CAR to specifically target glucuronoxylomannan (GXM) in the capsule of Cryptococcus species, called GXMR-CAR or GXMR-IgG4-28ζ. Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28ζ redirected T cells to target yeast and titan cell forms of Cryptococcus spp. Here, we replaced the IgG4-hinge and CD28-transmembrane domains from GXMR-CAR with a CD8α molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28ζ and GXMR-8-BBζ, respectively. Jurkat cells expressing GXMR-CAR containing CD8α as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28ζ and GXMR-8-BBζ induced high levels of IL-2 and up-regulation of CD69 expression in the presence of reference strains of C. neoformans and C. gattii. Moreover, GXMR-8-28ζ and GXMR-8-BBζ showed increased strength in response to incubation with clinical isolates of Cryptococcuss spp., and 4-1BB co-stimulatory domain triggered a more pronounced cellular activation. Dasatinib, a tyrosine kinase inhibitor, attenuated the GXMR-CAR signaling cascade's engagement in the presence or absence of its ligand. This study optimized novel second-generation GXMR-CARs containing the CD8-hinge/transmembrane domain that improved CAR expression, antigen recognition, and signal strength in T-cell activation.

Características clínicas, métodos diagnósticos y evolución de la criptococosis extrameníngea en personas viviendo con VIH / Clinical features, diagnosis methods and evolution in non meningeal cryptococcosis in people living with VIH

La criptococosis es una micosis grave que se manifiesta, en el 90% de los casos, como una meningoencefalitis, especialmente en las personas con VIH. El objetivo de este estudio es describir los casos de criptococosis extrameníngea en personas viviendo con VIH y conocer cuántas de estas padecen compromiso meníngeo concomitante. Además, determinar la relación con el título de antígeno polisacárido capsular de Cryptococcus en suero. Se realizó un estudio retrospectivo, observacional y analítico. Se incluyeron personas viviendo con VIH cuyo diagnóstico inicial de criptococosis se había realizado a partir de muestras extrameníngeas en el período comprendido entre 2012 y 2019. Los pacientes se dividieron en dos grupos. Grupo 1, pacientes sin compromiso meníngeo; Grupo 2, aquellos que finalmente tenían compromiso del SNC. De un total de 531 criptococosis registradas en ese período, se incluyeron 113 pacientes (21%), de los cuales en 58 se comprobó el compromiso meníngeo. No se observaron diferencias significativas en cuanto a la mortalidad entre ambos grupos.Ninguno de los pacientes con antigenemia por LFA (antígeno capsular en suero por inmunocromatografía) positiva, pero con antigenemia por aglutinación de partículas de látex (AL) negativa, tuvo compromiso meníngeo. Se observó que títulos de antígeno para Cryptococcus en suero por AL mayor o igual a 1/100 se correlacionaron con un aumento de 30 veces en la posibilidad de padecer meningitis. En todos los casos se debe descartar el compromiso del SNC. La AL sigue siendo una prueba útil y complementaria, debido a que en los casos con AL negativa no se observó compromiso meníngeo

Cryptococcosis is a serious mycosis that manifests itself, in 90% of cases, as meningoencephalitis, especially in AIDS patients. The objective of this study is to describe the extra-meningeal cases of cryptococcosis in people living with HIV and to know how many of them suffer from concomitant meningeal involvement. Also, to determine its relationship with the Cryptococcus capsular polysaccharide antigen titer in serum.A retrospective, observational and analytical study was carried out. HIV-positive patients whose initial diagnosis had been made from extrameningeal samples in the period between 2012 and 2019 were included. The patients were divided into 2 groups. Group 1: patients without meningeal involvement; group 2: those who finally had CNS involvement.Of a total of 531 cryptococcosis registered in this period, 113 patients (21%) were included, of whom meningeal involvement was confirmed in 58. No significant differences were observed in terms of mortality in both groups.None of the patients with positive LFA antigenemia (Capsular antigen detection by lateral Flow assay) but negative latex particle agglutination (LA) antigenemia had meningeal involvement. LFA was found to be highly sensitive and allows early diagnosis, but it does not replace other diagnostic procedures.Serum Cryptococcus antigen titers for by LA greater than or equal to 1/100 were found to correlate with a 30-fold increase in the likelihood of meningitis.In all cases, CNS involvement must be ruled out. LA continues to be a useful and complementary test, because in cases with negative LA, no meningeal involvement was observed

Inhibition of myeloid-derived suppressor cell arginase-1 production enhances T-cell-based immunotherapy against Cryptococcus neoformans infection.

Cryptococcosis is a potentially lethal disease that is primarily caused by the fungus Cryptococcus neoformans, treatment options for cryptococcosis are limited. Here, we show glucuronoxylomannan, the major polysaccharide component of C. neoformans, induces the recruitment of neutrophilic myeloid-derived suppressor cells in mice and patients with cryptococcosis. Depletion of neutrophilic myeloid-derived suppressor cells enhances host defense against C. neoformans infection. We identify C-type lectin receptor-2d recognizes glucuronoxylomannan to potentiate the immunosuppressive activity of neutrophilic myeloid-derived suppressor cells by initiating p38-mediated production of the enzyme arginase-1, which inhibits T-cell mediated antifungal responses. Notably, pharmacological inhibition of arginase-1 expression by a specific inhibitor of p38, SB202190, or an orally available receptor tyrosine kinase inhibitor, vandetanib, significantly enhances T-cell mediated antifungal responses against cryptococcosis. These data reveal a crucial suppressive role of neutrophilic myeloid-derived suppressor cells during cryptococcosis and highlight a promising immunotherapeutic application by inhibiting arginase-1 production to combat infectious diseases.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase

Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.(AU)

Opportunistic Invasive Fungal Infections Mimicking Progression of Non-Small-Cell Lung Cancer.

BACKGROUND: Many studies have shown that invasive pulmonary aspergillosis, cryptococcosis, and mucormycosis can mimic radiographic and clinical features of primary lung cancer. However, more research surveying the incidence and outcomes of these fungal infections among patients with a history of lung cancer is needed. The aim of this study was to describe the occurrence and clinical outcomes of opportunistic invasive fungal infections that can mimic tumors in non-small-cell lung cancer patients. PATIENTS AND METHODS: Patients seen at Stanford University Medical Center from January 1, 2007, to May 1, 2020, with pulmonary aspergillosis, cryptococcosis, or mucormycosis after non-small-cell lung cancer (NSCLC) diagnosis were reviewed. The European Organization for Research and Treatment of Cancer National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria was used to classify patients with evidence of proven or probable invasive fungal infection within our cohort. RESULTS: A total of 12 patients with proven or probable invasive mold infection (including 8 cases of aspergillosis) and 1 patient with proven cryptococcosis were identified, without any cases of mucormycosis. Of this cohort, 6 patients (46%) showed radiographic findings that were found to be most consistent with lung cancer by radiologists. Eight cases (62%) were suspected of cancer recurrence or progression by the treatment team on the basis of additional considerations of medical history and clinical symptoms. Most patients had active NSCLC or had a history of recurrence without active NSCLC at the time of fungal discovery (11 patients; 85%). Most patients died without full recovery (7 patients; 54%). CONCLUSIONS: Invasive pulmonary aspergillosis and cryptococcosis can often be mistaken as cancer recurrence or progression in patients with a history of NSCLC because of mimicking radiographic and clinical characteristics.

Panniculitis in a Woman With Opportunistic Pulmonary Coinfection by Pneumocystis jirovecii and Cryptococcus neoformans: 18F-FDG PET/CT Revealing the Infection and Assessing Treatment Response.

ABSTRACT: A 56-year-old woman, with history of psoriasis well controlled on ustekinumab, underwent 18F-FDG PET/CT to explore first onset of histologically proven skin panniculitis of unknown origin. PET/CT showed high uptake in panniculitis lesions in limbs and in a lung opacity suggestive of pneumonia. Based on PET/CT findings, a bronchoalveolar lavage revealed pulmonary coinfection by Pneumocystis jirovecii and Cryptococcus neoformans. Thus, hematogenous dissemination of infection was suspected as etiology of panniculitis. She was treated with fluconazole and trimethoprim-sulfamethoxazole, leading to total resolution of skin lesions. Posttherapeutic PET/CT showed complete metabolic response of skin and pulmonary lesions.

The interplay of phenotype and genotype in Cryptococcus neoformans disease.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis primarily in immunocompromised individuals. In order to survive and proliferate during infection, C. neoformans must adapt to a variety of stresses it encounters within the host. Patient outcome depends on the interaction between the pathogen and the host. Understanding the mechanisms that C. neoformans uses to facilitate adaptation to the host and promote pathogenesis is necessary to better predict disease severity and establish proper treatment. Several virulence phenotypes have been characterized in C. neoformans, but the field still lacks a complete understanding of how genotype and phenotype contribute to clinical outcome. Furthermore, while it is known that C. neoformans genotype impacts patient outcome, the mechanisms remain unknown. This lack of understanding may be due to the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates during infection. In this review, we summarize the current understanding of how the various genotypes and phenotypes observed in C. neoformans correlate with human disease progression in the context of patient outcome and recurrence. We also postulate the mechanisms underlying the genetic and phenotypic changes that occur in vivo to promote rapid adaptation in the host.

Fungal Co-infections Associated with Global COVID-19 Pandemic: A Clinical and Diagnostic Perspective from China.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been sweeping across the globe. Based on a retrospective analysis of SARS and influenza data from China and worldwide, we surmise that the fungal co-infections associated with global COVID-19 might be missed or misdiagnosed. Although there are few publications, COVID-19 patients, especially severely ill or immunocompromised, have a higher probability of suffering from invasive mycoses. Aspergillus and Candida infections in COVID-19 patients will require early detection by a comprehensive diagnostic intervention (histopathology, direct microscopic examination, culture, (1,3)-ß-D-glucan, galactomannan, and PCR-based assays) to ensure effective treatments. We suggest it is prudent to assess the risk factors, the types of invasive mycosis, the strengths and limitations of diagnostic methods, clinical settings, and the need for standard or individualized treatment in COVID-19 patients. We provide a clinical flow diagram to assist the clinicians and laboratory experts in the management of aspergillosis, candidiasis, mucormycosis, or cryptococcosis as co-morbidities in COVID-19 patients.

Combatting the evolution of antifungal resistance in Cryptococcus neoformans.

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.

Clinical analysis in immunocompetent and immunocompromised patients with pulmonary cryptococcosis in western China.

Cryptococcosis is a systemic infection and it may occur in immunocompromised and immunocompetent hosts. In order to better understand the clinical characteristics of patients with PC in different immune status, we retrospectively investigated the clinical, radiological, and treatment profiles of immunocompetent and immunocompromised patients with PC during a 10-year period (2008-2017). As a result, out of 136 patients, 94 (69.1%) were immunocompromised hosts. For the PC patients without CNS involvement, higher percentage of immunocompetent patients (39.5%, 15/38) had asymptomatic presentation than immunocompromised patients (6.3%, 3/48) (P < 0.05). Multiple pulmonary nodules (72.7%, 56/77), ground-glass attenuation/interstitial changes (94.4%, 17/18) and cavitation (88.6%, 31/35) were significantly frequent in immunocompromised patients (P < 0.05). A total of 47 patients were misdiagnosed as tuberculosis or tumors based on CT signs. PC was likely to be misdiagnosed as tuberculosis in immunocompromised patients (88.2%, 15/17), and tumor was more likely to be considered in immunocompetent patients (43.3%, 13/30). Immunocompetent patients accounted for 80% (24/30) of patients with definite diagnosis on surgical lung biopsy. Fluconazole monotherapy can achieve good clinical outcome in most PC patients without central nervous system (CNS) involvement (91.5%, 54/59). After 3 months of treatment, 92.7% (38/41) patients have improved imaging findings. In conclusion, PC has diverse imaging manifestations and it is easily misdiagnosed. Lobectomy should be carefully selected in immunocompetent patients with a single lung lesion. Fluconazole monotherapy is preferred for PC patients without CNS involvement.

An unusual cause for rib osteomyelitis in the tropics: Cryptococcal osteomyelitis.

The more common manifestations of cryptococcal infections are restricted to the central nervous system and lungs. We report an unusual case of fungal osteomyelitis due to Cryptococcus. The patient was a young man who had been adequately treated for pulmonary tuberculosis three years prior. Three months before, he sustained a minor road-traffic accident with only minor abrasions. He presented with subacute chest pain of 15 days' duration and was found to have radiological evidence of a lytic lesion of the fifth rib. Given prior tuberculosis, he was thought to have a relapse of disease with tuberculous osteomyelitis. Surprisingly, a biopsy revealed evidence of fungal osteomyelitis with Cryptococcus. An evaluation for primary immunodeficiency revealed low CD4 cell counts with undetectable serum IgA and IgM levels. Genetic sequencing proved a genetic mutation consistent with primary T-cell immunodeficiency. The patient responded well to treatment and is asymptomatic on follow-up.

Cryptococcal pericarditis in a heart transplant recipient.

We present a case of Cryptococcus neoformans pericarditis in a cardiac transplant recipient. This article reviews the diagnosis, treatment, and complications of cryptococcosis specifically in transplant patients. While pericarditis is a rare manifestation of Cryptococcus infection, this case highlights that cryptococcosis should be considered in the differential diagnosis for solid organ transplant and immunocompromised patients presenting with pericardial effusions.

T11TS immunotherapy potentiates the repressed calcineurin-NFAT signalling pathway of T cells in Cryptococcus neoformans infected rats: a cue towards T-cell activation for antifungal immunity.

AIMS: To examine the modulation of the interacting partners of the calcineurin (CaN)-NFAT pathway in T cells during Cryptococcus neoformans fungal infection and post-T11TS immunotherapy. METHODS AND RESULTS: Wistar rats were infected with C. neoformans and followed by immunotherapy with immune-potentiator T11TS. T cells were analysed by flow cytometry, immunoblotting and nuclear translocation study. The signalling proteins LCK, FYN, LAT, PLCγ1 and CaN in T cells were regulated by C. neoformans infection resulting in reduced nuclear translocation of NFAT and IL-2 expression. Following T11TS immunotherapy, the expressions of the above-mentioned proteins were boosted and thus resulting in the clearance of C. neoformans from lung and spleen. CONCLUSIONS: The precise mechanism of suppression of the T-cell function by C. neoformans is still unknown. Previously, we have shown that T11TS positively regulates the function of T cells to abrogate glioma and other immunosuppressive conditions. T11TS immunotherapy increased the expression of the above signalling partners of the CaN-NFAT pathway in T cells and improved nuclear retention of NFAT. As a result, an increased IL-2 expression leads to activation and proliferation of T cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results demonstrate the role of T11TS in restoring the CaN-NFAT signalling pathway in T cells. It identifies T11TS as an immunotherapeutic agent with potential clinical outcomes to counteract C. neoformans infection.

Vaccines and Protective Immune Memory against Cryptococcosis.

Cryptococcosis is a potentially lethal disease caused by fungal pathogens including Cryptococcus neoformans and Cryptococcus gattii species complex. These fungal pathogens live in the environment and are associated with certain tree species and bird droppings. This infectious disease is not contagious, and healthy individuals may contract cryptococcal infections by inhaling the airborne pathogens from the environment. Although cleaning a contaminated environment is a feasible approach to control environmental fungal pathogens, prophylactic immunization is also considered a promising method to regulate cryptococcal infections. We review the history of the development of cryptococcal vaccines, vaccine components, and the various forms of immune memory induced by cryptococcal vaccines.

Are macrophages the heroes or villains during cryptococcosis?

Cryptococcus infections represents a major healthcare burden, with over 200,000 cases globally a year and even with treatment, mortality remains as high as 80%. There is a clear need for new classes of treatment, especially with the global threat of antifungal resistance. Several groups are investigating the potential of immunotherapy - circumventing many of the issues with current treatments. Macrophages are a cell type known to be heavily associated with cryptococcal infection, from the innate immune response through to the later stage chronic adaptive response - making these an ideal target for manipulation. However, it is currently debated whether macrophage activity is positive or negative for host outcomes. Here, we discuss the current literature surrounding the role of macrophages during Cryptococcus infection, and makes cases for and against macrophage enhancement. Finally, we discuss which pressing questions in the field still remain that require answers in order to safely design an immunotherapeutic with high efficacy.

TNF-α-Producing Cryptococcus neoformans Exerts Protective Effects on Host Defenses in Murine Pulmonary Cryptococcosis.

Tumor necrosis factor alpha (TNF-α) plays a critical role in the control of cryptococcal infection, and its insufficiency promotes cryptococcal persistence. To explore the therapeutic potential of TNF-α supplementation as a booster of host anti-cryptococcal responses, we engineered a C. neoformans strain expressing murine TNF-α. Using a murine model of pulmonary cryptococcosis, we demonstrated that TNF-α-producing C. neoformans strain enhances protective elements of host response including preferential T-cell accumulation and improved Th1/Th2 cytokine balance, diminished pulmonary eosinophilia and alternative activation of lung macrophages at the adaptive phase of infection compared to wild type strain-infected mice. Furthermore, TNF-α expression by C. neoformans enhanced the fungicidal activity of macrophages in vitro. Finally, mice infected with the TNF-α-producing C. neoformans strain showed improved fungal control and considerably prolonged survival compared to wild type strain-infected mice, but could not induce sterilizing immunity. Taken together, our results support that TNF-α expression by an engineered C. neoformans strain while insufficient to drive complete immune protection, strongly enhanced protective responses during primary cryptococcal infection.

Clostridioides difficile-related toxic megacolon after Cryptococcus neoformans cellulitis: A complex of two rare infections in an immunocompromised host.

A 76-year-old Japanese woman was admitted due to uncontrolled cellulitis of the right lower leg. She had deep vein thrombosis on the right limb. Moreover, she had a long history of rheumatoid arthritis treated with corticosteroids. Skin biopsy and lumbar puncture were performed to diagnose disseminated cryptococcosis. She was administered antifungal agents (liposomal amphotericin B and 5-fluorocytosine). On treatment day 14, debridement was performed, and cryptococcosis was controlled. However, she developed toxic megacolon due to Clostridioides difficile infection (CDI). On day 32, she was transferred to the intensive care unit due to severe acidosis and acute kidney injury secondary to CDI-related toxic megacolon. Vancomycin, metronidazole, and tigecycline were administered for treatment of CDI. After several weeks of intensive care, toxic megacolon was improved, but renal replacement therapy was discontinued according to the patient's will. On day 73, she died of renal failure. We experienced a complex of rare diseases, Cryptococcus neoformans cellulitis and Clostridioides difficile-related toxic megacolon. Both diseases were presumed to be the result of corticosteroid and methotrexate use. Hence, careful monitoring is required when treating immunocompromised hosts to reduce the risk of developing complications.