Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.Funding for this study was provided by National Institutes of Health grants EY08538 and GM34107 (E. Rodriguez-Boulan); EY027038 (R.F. Mullins); 1R21CA224391-01A1 (J.H. Zippin); and 1R01CA194547, 1U24CA210989, and P50CA211024 (O. Elemento); National Cancer Institute grant R01CA192176 and cancer center support grant P30 CA008748-48 (A.L. Joyner); Comunidad Autónoma de Madrid grant 2017-T1/BMD-5247 (I. Benedicto); Agencia Nacional Argentina de Promoción Cient´ıfica y Tecnológica grant PICT 2014-3687 and Fundación Sales (G.A. Rabinovich); a Pew Latin American Fellowship (G.L. Lehmann); Calder Research Scholar Award Vitiligo/Pigment Cell Disorders (J.H. Zippin); Starr Foundation Tri-Institutional Stem Cell Initiative award 2013-028; NYSTEM contract C32596GG; and Research to Prevent Blindness and Dyson Foundation departmental grants. The CNIC is supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Antibody- and T Cell-Dependent Responses Elicited by a SARS-CoV-2 Adenoviral-Based Vaccine in a Socially Vulnerable Cohort of Elderly Individuals

Background: In spite of compelling evidence demonstrating safety and immunogenicity of adenoviral-based SARS-CoV-2 vaccines in the general population, its effects in socially vulnerable elderly individuals are poorly understood. Here we aimed to investigate the efficacy of two doses of combined vector vaccine, the Gam-COVID-Vac (Sputnik-V vaccine), at 14, 42, and 180 days after immunization, in a nursing home for underprivileged population and homeless individuals. Methods: A phase 3, open-label clinical trial involving administration of two adenoviral vectors (Ad26-Ad5) vaccine, in elderly individuals over the ages of 60 years was performed. SARS-CoV-2 Spike RBD-specific IgG antibodies at days 21-, 42- and 180 post-vaccination was analyzed in sera of individuals receiving two doses of the Sputnik-V vaccine with an interval of 21 days. SARS-CoV-2-specific CD8+ T cell responses, measured by intracellular tumor necrosis factor (TNF) was determined by flow cytometry following antigen-specific cultures. Results: A total of 72 elderly adults with a mean age of 72.6 ± 9.5 years-old was selected after applying the inclusion criteria, all corresponding to an underprivileged population. Two-doses vaccination with Sputnik-V vaccine elicited an antibody-mediated immune response (revealed by quantitative detection of SARS-CoV-2-specific IgG antibodies, CMIA) 70% at day 21, 90% at day 42, and 66.1% at day 180. Fully vaccinated individuals had robust SARS-CoV-2-specific T cell responses, evidenced by TNF production in CD4+ and CD8+ T cells in all time periods analyzed. Conclusion: Six months after receipt of the second dose of the Gam-COVID-Vac vaccine, SARS-CoV-2-specific IgG levels declined substantially among the tested population, whereas CD4+ and CD8+ T-cell-mediated immunity remained at high levels. These data suggest that two doses of combined adenoviral-based vaccine elicits a considerable level of SARS-CoV-2 immune responses in elderly individuals, highlighting its safety and immunogenicity in this highly vulnerable population

Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation

Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+), and bicarbonate (HCO3 −). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+]i) in individual live sperm. An increase in [Ca2+]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+]i. In addition, it was determined that the capacitation-associated [Ca2+]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+]i. Surprisingly, a minimum increase in [Ca2+]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+.Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: D'alotto Moreno, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Martín Hidalgo, David. University of Massachussets; Estados UnidosFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Puga Molina, Lis del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Schiavi Ehrehaus, Liza. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gilio, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin