An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Índices de oxigenación como predictores de ventilación mecánica en neumonía a 2600 metros de altitud / Oxygen indexes as predictors of mechanical ventilation in pneumonia at 2600 meters above sea level

Resumen Antecedentes: los valores de la diferencia alveolo arterial de oxígeno D(A-a)O2 y de la relación presión alveolar de oxígeno y fracción inspirada de oxígeno (PaO2/FiO2), son pobremente conocidos a gran altitud para predecir ventilación mecánica (VM) en pacientes con neumonía adquirida en comunidad (NAC) mayores de 65 años. Objetivo: conocer los valores de D(A-a)O2 y PaO2/FiO2 en pacientes con NAC que requirieron soporte ventilatorio. Métodos: estudio de cohorte prospectivo donde se obtuvo la D(A-a)O2 y PaO2/FiO2 de los gases arteriales de ingreso a urgencias, con cálculo de sensibilidad (S), especificidad (E), valor predictivo positivo (VPP), valor predictivo negativo VPN) y área bajo la curva ROC para el requerimiento de VM en las primeras 72 horas. Resultados: se siguieron 247 pacientes, 37 (15%) requirieron VM, no se encontraron diferencias en edad, género, y comorbilidades entre los grupos de VM y no VM. El área bajo la curva ROC para D(A-a) O2 como predictor de VM fue de 0.84 (IC95%:0.77-0.92), para la PaO2/FiO2 de 0.85 (IC 5%: 0.78-0.92) (p<0.0001). Para una D(A-a)O2 en 55 se obtuvo una sensibilidad para predecir VM en 70.27%, especificidad 86.19%, VPP: 47%, VPN: 94%, razón de verosimilitud positiva (LR+): 5.1, razón de verosimilitud negativa (LR-): 0.3. Una PaO2/FiO2 de 180 tiene una sensibilidad para predecir VM de: 86.65%, especificidad: 70.27%, VPP: 34%, VPN: 97%, LR+: 2.9, LR-: 0.2. La mortalidad global fue 3.2%. Conclusión: los valores de D(A-a)O2 y PaO2/FiO2 se relacionan con el requerimiento de VM en pacientes mayores de 65 años con NAC. (Acta Med Colomb 2016; 41: 169-175).

Abstract Background: the values of the difference of alveolar arterial oxygen D(A-a)O2 and ratio of the alveolar oxygen pressure and fraction of inspired oxygen (PaO2/FiO2) are poorly known at high altitude to predict mechanical ventilation (MV) in patients over 65 years with community-acquired pneumonia (CAP). Objective: to know the values of D(A-a)O2 and PaO2/FiO2 in CAP patients requiring ventilatory support. Methods: prospective cohort study where D(A-a)O2 y PaO2/FiO2 were obtained from arterial blood gases at entrance to the emergency room, with calculation of sensitivity (S), specificity (E), positive predictive value (PPV), negative predictive value (NPP) and area under the ROC curve for MV requirement within the first 72 hours. Results: 247 patients were followed; 37 (15%) required MV. No differences were found in age, gender and comorbidities between the groups of MV and no MV. The area under the ROC curve for D(A-a) O2 as a predictor of MV was 0.84 (95% CI: 0.77 to 0.92), for the la PaO2/FiO2 of 0.85 (95% CI: 0.78 to 0.92) (p <0.0001). For a D(A-a)O2 in 55 patients was obtained a sensibility to predict MV in 70.27%, specificity 86.19%, PPV 47%, NPV 94%, positive likelihood ratio (LR +): 5.1, negative likelihood ratio (LR -): 0.3. A PaO2/FiO2 of 180 has a sensitivity to predict MV of 86.65%, specificity: 70.27%, PPV 34%, NPV 97%, LR +: 2.9, LR: 0.2. Overall mortality was 3.2%. Conclusion: the values of D(A-a)O2 and PaO2/FiO2 relate to the requirement of MV in patients older than 65 with CAP. (Acta Med Colomb 2016; 41: 169-175).