Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.

Target lysis by cholesterol extraction is a rate limiting step in the resolution of phagolysosomes.

The ongoing phagocytic activity of macrophages necessitates an extraordinary capacity to digest and resolve incoming material. While the initial steps leading to the formation of a terminal phagolysosome are well studied, much less is known about the later stages of this process, namely the degradation and resolution of the phagolysosomal contents. We report that the degradation of targets such as splenocytes and erythrocytes by phagolysosomes occurs in a stepwise fashion, requiring lysis of their plasmalemmal bilayer as an essential initial step. This is achieved by the direct extraction of cholesterol facilitated by Niemann-Pick protein type C2 (NPC2), which in turn hands off cholesterol to NPC1 for export from the phagolysosome. The removal of cholesterol ulimately destabilizes and permeabilizes the membrane of the phagocytic target, allowing access of hydrolases to its internal compartments. In contrast, we found that saposins, which activate the hydrolysis of sphingolipids, are required for lysosomal tubulation, yet are dispensable for the resolution of targets by macrophages. The extraction of cholesterol by NPC2 is therefore envisaged as rate-limiting in the clearance of membrane-bound targets such as apoptotic cells. Selective cholesterol removal appears to be a primary mechanism that enables professional phagocytes to distinguish the target membrane from the phagolysosomal membrane and may be conserved in the resolution of autolysosomes.

Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.

The Scribble Complex PDZ Proteins in Immune Cell Polarities.

hScrib and hDlg belong to the PDZ family of proteins. Since the identification of these highly phylogenetically conserved scaffolds, an increasing amount of experiments has elucidated the roles of hScrib and hDlg in a variety of cell functions. Remarkably, their participation during the establishment of polarity in epithelial cells is well documented. Although the role of both proteins in the immune system is scantly known, it has become a growing field of investigation. Here, we summarize the interactions and functions of hScrib and hDlg1, which participate in diverse functions involving cell polarization in immune cells, and discuss their relevance in the immune cell biology. The fundamental role of hScrib and hDlg1 during the establishment of the immunological synapse, hence T cell activation, and the recently described role of hScrib in reactive oxygen species production in macrophages and of hDlg1 in cytokine production by dendritic cells highlight the importance of both proteins in immune cell biology. The expression of these proteins in other leukocytes can be anticipated and needs to be confirmed. Due to their multiple interaction domains, there is a wide range of possible interactions of hScrib and hDlg1 that remains to be explored in the immune system.

Scrib and Dlg1 polarity proteins regulate Ag presentation in human dendritic cells.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Identification of conserved peptides containing B-cell epitopes of Babesia bovis AMA-1 and their potential as diagnostics candidates.

The apical membrane antigen 1 (AMA-1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA-1 plays an essential role for parasite invasion to target cells. It has been reported that AMA-1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B-cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B-cell epitopes from the extracellular region of Babesia bovis AMA-1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra-erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA-1 from B. bovis has conserved B-cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed.

PDZ proteins are expressed and regulated in antigen-presenting cells and are targets of influenza A virus.

In this work, we identified the expression, regulation, and viral targeting of Scribble and Dlg1 in antigen-presenting cells. Scribble and Dlg1 belong to the family of PDZ (postsynaptic density (PSD95), disc large (Dlg), and zonula occludens (ZO-1)) proteins involved in cell polarity. The relevance of PDZ proteins in cellular functions is reinforced by the fact that many viruses interfere with host PDZ-dependent interactions affecting cellular mechanisms thus favoring viral replication. The functions of Scribble and Dlg have been widely studied in polarized cells such as epithelial and neuron cells. However, within the cells of the immune system, their functions have been described only in T and B lymphocytes. Here we demonstrated that Scribble and Dlg1 are differentially expressed during antigen-presenting cell differentiation and dendritic cell maturation. While both Scribble and Dlg1 seem to participate in distinct dendritic cell functions, both are targeted by the viral protein NS1 of influenza A in a PDZ-dependent manner in dendritic cells. Our findings suggest that these proteins might be involved in the mechanisms of innate immunity and/or antigen processing and presentation that can be hijacked by viral pathogens.

Bromuro de rocuronio: una nueva alternativa como relajante muscular: experiencias en el Centro Quirúrgico del Hospital Arzobispo Loayza / Rocuronium bromide: a new alternative how muscular relaxant: experiences in the Surgical Center of Hospital Arzobispo Loayza

En el presente trabajo se realiza un estudio prospectivo, donde se evalúa las características de un nuevo relajante muscular no despolarizante de acción intermedia, que presenta un tiempo de latencia y desarrollo del bloqueo neuromuscular muy corto, que permite condiciones de intubación excelentes a los 60 segundos y un mantenimiento de la relajación muscular transoperatoria satisfactoria con una adecuada estabilidad cardiovascular. El estudio se realizó en 50 pacientes del Hospital General Nacional Arzobispo Loayza, que fueron sometidos a diversos tipos de cirugía: en las cuales se usó anestesia general inhalatora con sevofluorane, usándose para la intubación endotraqueal y el mantenimiento de la relajación muscular Bromuro de Rocuronio. El estudio comprendió 50 pacientes de ambos sexos cuyas edades fluctuaban entre 24 y 68 años de edad, ASA I y II, obteniéndose estabilidad hemodinámica durante la intubación endotraqueal y el mantenimiento anastésico llegándose a concluir que este nuevo relajante muscular es una buena alternativa para ser utilizado durante la intubación endotraqueal y en la relajación muscular transoperatoria, proporcionando seguridad y una recuperación satisfactoria, sin producir los efectos propios de los relajantes despolarizantes.

Uso de xilocaina-fentanyl por vía epidural: experiencias en el Centro Quirúrgico del Hospital Arzobispo Loayza / Use of xilocaine-fentanyl by epidural via: Experiences in the Surgical Center of Hospital Arzobispo Loayza

Se proyectó la utilización de la técnica epidural, que asocia Xilocaína al 2 por ciento con epinefrina y Fentanil con la finalidad de obtener una alternativa útil de bajo costo y sin mayores complicaciones; y que permitiera la prolongación del efecto analgésico de dicho procedimiento. Con tal finalidad se seleccionaron 106 pacientes que iban a ser sometidos a intervención quirúrgica extraabdominal o que no iban a requerir el uso de relajantes musculares. Se elaboró un protocolo de trabajo general, que consignaba datos generales, premedicación anestésica y la técnica anestésica. La totalidad de los pacientes fueron ASA I - III. La gran mayoría de los pacientes fueron de sexo femenino y sus edades estaban entre los 18 y los 74 años. La premedicación anestésica fue de 10 mgr, de Diazepan y 0.5 mgr. de Sulfato de Atropina, 30 minutos antes de la intervención, previa a la punción lumbar. La posición del paciente, para la aplicación de la anestesia epidural fue sentada en todos los casos, siendo el nivel de punción L1-L2 ó L2-L3, utilizando para tal caso una aguja Touhy número 17 ó 18, utilizando como artificio la pérdida de la resistencia al llegar al espacio peridural. No hubo perforación de duramadre en ningún caso. La dosis utilizada fue de 360 mgr. de Xilocaína 2 por ciento CE. y 100 ugr. de Fentanyl. Se evaluó el nivel de bloqueo a los 5 minutos posteriores a la punción lumbar, llevándose el control tanto de la duración del acto quirúrgico, como en el tiempo de duración de la analgesia. La mayoria de las operaciones tuvieron una duración por encima de las 2 horas. El tiempo de duración de la analgesia en la gran mayoría de los casos fue superior a las 2 horas, siendo el promedio obtenido en el presente estudio el de 2 horas con 24 minutos, permitiendo con ello que el paciente obtenga analgesia post-operatoria en todos los casos...

Manejo anestésico de miastenia gravis: a propósito de un caso / Management anesthesic of miastenia gravis: purpose of a case

Se utilizó la presente técnica anestésica en una paciente de 28 años de edad, sexo femenino, ASA I, diagnosticada de Miastenia Gravis, que fue sometida a Timectomía mediante la utilización de anestesia general inhalatoria, utilizando en la inducción anestésica la asociación de Fentanylo-Midazolam y Barbitúrico, ningún relajante muscular y Sevoflurante como anestésico de mantenimiento. Comportándose en el Trans-operatorio la paciente con gran estabilidad hemodinámica y respiratoria, habiendo presentado una rápida y buena recuperación, concluyéndose que dicha técnica es una alternativa para ser utilizada en este tipo de pacientes.

Anestesia general en cirugía laparoscópica / General anesthesia in laparoscopic surgery

El presente trabajo consistió en evaluar una nueva técnica anestésica para ser utilizada en cirugía laparoscópica. Se estudiaron 50 pacientes de ambos sexos, con edades entre 19 y 67 años, ASA I-II, que fueron sometidos a anestesia general para cirugía abdominal y ginecológica, usando la asociación Fentanyl-Midazolan, barbitúrico en la inducción anestésica, bromuro de pipecuronio como relajante muscular, isoflurane como anestésico inhalatorio de mantenimiento. Obteniéndose gran estabilidad hemodinámica y respiratoria como resultado satisfactorio y concluyendo que dicha técnica es una buena alternativa a usarse con el objetivo de brindar buenas condiciones quirúrgicas; seguridad y bienestar transoperatorios, como también una recuperación postoperatoria rápida de la anestesia.