A high-dimensional platform for observing neutrophil-parasite interactions.

Diarrheal diseases with infectious etiology remain a major cause of death globally, particularly in low-income countries. Entamoeba histolytica is a pathogenic protozoan parasite that is the causative agent of amebiasis. Amebiasis has a wide presentation in clinical severity with many factors, including the bacterial microbiota, contributing to this variation. The innate immune response also plays a critical role in regulating the severity of E. histolytica infection, with neutrophils reported to have a protective role. Despite this, the precise mechanism of how neutrophils mediate amebic killing is poorly understood. Thus, modern platforms that allow for inquiry of granulocyte-ameba interactions will increase our understanding of this disease. Herein, we describe an assay for neutrophil killing of E. histolytica by utilizing high-dimensional spectral flow cytometry. Neutrophils were isolated from wild-type 5-week-old C57BL/6 mice and co-cultured with E. histolytica at various multiplicity of infections (MOIs). After co-culture, neutrophils and E. histolytica were stained for spectral flow cytometry. Cell populations were identified using surface markers and fluorescence minus one (FMO) controls. We have previously shown that animals colonized with a component of the human microbiota, Clostridium scindens, were protected from E. histolytica. This protection was associated with elevated neutrophil count. Here, we explored amebic killing capacity and observed that neutrophils from animals with C. scindens possessed heightened amebic killing compared with controls. Thus, this study establishes a novel platform that can provide an in-depth analysis of granulocyte-parasite interactions in various contexts, including during alteration of the intestinal microbiota.IMPORTANCEThe tools for studying host immune cell-E. histolytica interactions are limited. Factors, such as parasite heterogeneity, infectivity, and difficulties with culture systems and animal models, make interrogation of these interactions challenging. Thus, Entamoeba researchers can benefit from next-generation models that allow for the analysis of both host and parasite cells. Here, we demonstrate the use of a novel platform that allows for the determination of parasite-host cell interactions and customizable high-dimensional phenotyping of both populations. Indeed, spectral flow cytometry can approach >40 markers on a single panel and can be paired with custom-developed parasite antibodies that can be conjugated to fluorochromes via commercially available kits. This platform affords researchers the capability to test highly precise hypotheses regarding host-parasite interactions.

Secondary bile acids function through the vitamin D receptor in myeloid progenitors to promote myelopoiesis.

Metabolic products of the microbiota can alter hematopoiesis. However, the contribution and site of action of bile acids is poorly understood. Here, we demonstrate that the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), increase bone marrow myelopoiesis. Treatment of bone marrow cells with DCA and LCA preferentially expanded immunophenotypic and functional colony-forming unit-granulocyte and macrophage (CFU-GM) granulocyte-monocyte progenitors (GMPs). DCA treatment of sorted hematopoietic stem and progenitor cells (HSPCs) increased CFU-GMs, indicating that direct exposure of HSPCs to DCA sufficed to increase GMPs. The vitamin D receptor (VDR) was required for the DCA-induced increase in CFU-GMs and GMPs. Single-cell RNA sequencing revealed that DCA significantly upregulated genes associated with myeloid differentiation and proliferation in GMPs. The action of DCA on HSPCs to expand GMPs in a VDR-dependent manner suggests microbiome-host interactions could directly affect bone marrow hematopoiesis and potentially the severity of infectious and inflammatory disease.

The IL-33-ILC2 pathway protects from amebic colitis.

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Vogt-Koyanagi-Harada Disease Following COVID-19 Infection.

A 29-year-old female presented to the emergency clinic with gradual visual disturbance in both eyes for 15 days duration, accompanied by bilateral tinnitus, and ocular pain that increased with ocular movements. One month prior to presentation, the patient had tested positive for severe acute respiratory syndrome coronavirus-2 but without complications. Visual acuity was 20/100 in the right eye and 20/300 in the left eye. Funduscopy demonstrated optic nerve swelling, radial nerve fiber striation disruption, and bilateral retinal folds. Optical coherence tomography showed serous (bacillary) retinal detachment and multifocal areas of hyper-reflective changes in the inner and outer plexiform layer with inner nuclear layer thickening and disruption of the interdigitation zone bilaterally. We present a case of incomplete Vogt-Koyanagi-Harada disease following COVID-19 infection.

Cirugía filtrante ajustable: nueva técnica antiglaucomatosa / Adjustable filtering surgery: technique new in antiglaucomatous surgery

Ofrecer una técnica quirúrgica en pacientes con glaucoma de ángulo abierto, que hemos denominado cirugía filtrante ajustable, de fácil realización, cuyo objetivo fundamental es impedir el cierre de la fístula por el proceso de fibrosis de la herida. Seis pacientes entre 44 y 75 años, con diagnóstico de glaucoma de ángulo abierto avanzado no controlado con terapia máxima, fueron sometidos a esta técnica quirúrgica una vez obtenido su consentimiento, con excavaciones entre 0,6 y 0,7 y presión intraocular mínima de 28 mmHg y máxima de 32mmHg. En 5 de 6 pacientes se obtuvieron niveles de presión intraocular entre 14 y 17 mmHg. Cuando se movilizaba el flap se obtenía una disminución de presión intraocular de 3-4 mmHg. En 1 paciente el nivel de presión persistió elevado y se añadió terapia médica. El mecanismo de acción de nuestro procedimiento consiste en interrumpir el ciclo de formación de la fibrosis subconjuntival mediante un efecto mecánico provocado por la tracción del flap en el lecho filtrante, durante los días de máxima proliferación fibroblástica, pero se necesitan futuros estudios clínicos e histopatológicos para determinar la eficiencia de este procedimiento