Francisco Otaizo-Carrasquero

Background: Despite the efficacy of therapeutics and vaccines in reducing risk of severe coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections continue to occur and rebound symptoms after initial improvement have been well described. The mechanisms driving COVID-19 rebound remain unclear. Methods: Critical inflammatory responses were assessed by Imagestream, flow cytometry, and single-cell RNA sequencing in peripheral blood mononuclear cells from vaccinated individuals presenting with early (3 days postinfection, n = 6) or late (8 days postinfection, n = 6) acute symptoms, and clinical rebound (16 days postinfection, n = 8), as well as healthy volunteers (HV, n = 7). Results: An overall decline in key inflammatory responses was observed in groups with longer time from symptom onset when compared to early acute infection. However, RNA sequencing revealed a partial resurgence of inflammatory, stress, and antiviral responses during clinical rebound, with a unique cluster of monocytes with greater activation of antigen presentation pathways and type I interferon signaling. Monocytes from patients with rebound COVID-19 also showed elevated inflammasome activation when compared to HV, along with an accumulation of oxidized lipids and decreased levels of glutathione, both hallmarks of the oxidative stress response. Inflammatory measurements positively associated with serum SARS-CoV-2 nucleocapsid antigen and inversely correlated with adaptive immune responses. Conclusions: Our findings potentially reflect viral reappearance posttreatment in participants with clinical rebound and outline mechanisms by which rebound symptoms are typically milder than during acute presentations. Extending the antiviral treatment period and/or targeting inflammatory pathways could potentially prevent virological rebound or help mitigate associated symptoms. Clinical Trials Registration: NCT04401436.

Background: Cerebral venous sinus thrombosis (CVST), though a recognized complication of cryptococcal meningitis (CM), has been rarely reported. This study investigates the clinical features of CVST in a retrospective cohort of previously healthy, non-HIV CM patients. Methods: We reviewed medical records of 89 immunocompetent CM patients admitted between January 2005 and April 2024. CVST incidence, clinical course, neuroimaging, and laboratory data were analyzed. Genetic analysis was performed to detect thrombophilia-associated variants. To explore thrombosis-related gene expression during neuroinflammation, cerebrospinal fluid (CSF) cells collected at the time of cryptococcal post-infectious inflammatory response syndrome (cPIIRS) diagnosis were subjected to single-cell RNA sequencing. Results: CVST occurred in 5.6% (CI 1.9%–12.6%) of CM patients, a rate comparable to bacterial CNS infections. No known pathogenic variants in known thrombophilia-associated genes were identified. However, 44 genes previously linked to thrombosis showed elevated expression—defined as ≥150 single-cell sequencing reads—in a cPIIRS patient. Conclusions: CVST is a potentially treatable complication of CM. MRI and MRV of the brain are valuable diagnostic tools. The absence of thrombophilia mutations suggests that neurologic infection itself contributes to thrombotic risk. Notably, CSF gene expression patterns may serve as biomarkers for CVST susceptibility. Future case-control studies may validate these findings and uncover genetic risk factors contributing to CVST.

Therapies to reduce neuroinflammation following resolution of acute central nervous system (CNS) infections are urgently needed, particularly for patients with non–HIV-associated cryptococcal meningoencephalitis complicated by a postinfectious inflammatory response syndrome (cPIIRS). To identify druggable targets in cPIIRS, patient cerebral spinal fluid samples underwent transcriptional analysis, revealing a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway dominance in neuroinflammatory gene signatures. MurinecPIIRS models recapitulated this pathway predominance and treatment with the JAK inhibitor ruxolitinib, confirmed a mechanistic requirement for this pathway in disease pathology. Ruxolitinib treatment improved markers of neuronal damage, reduced activated T cell and myeloid cells, and improved weight. On the basis of these findings, we conducted a first-in-human ruxolitinib treatment of patients with cPIIRS (NCT00001352). Ruxolitinib treatment of six patients led to demonstrated tolerability, reductions in inflammatory biomarkers and activated immune cells, and improved brain imaging. These results advocate for pathway-instructed therapeutics in neuroinflammatory diseases and endorse JAK inhibitors in further clinical studies of cPIIRS.

Human Th17/type 17 cells express the chemokine receptor CCR6, but the functions of CCR6 and other chemokine receptors in human type 17 Th cell extravasation have not been fully delineated. Here we show that human peripheral blood CD4⁺CCR6⁺ T cells co-expressing CCR2 have a pathogenic Th17 signature, can produce inflammatory cytokines without T cell receptor activation, and show enhanced expression of pathogenicity-associated and activation-associated genes in the cerebrospinal fluid of patients with multiple sclerosis as compared to controls. In flow chambers with activated endothelial cell (EC) monolayers, CD4⁺CCR6⁺CCR2⁺ T cells are efficient at transendothelial migration (TEM). Ligands for CCR5, CCR6 and CXCR3 localize to EC surfaces and mediate only arrest, whereas CCR2 ligands fail to bind well to ECs and mediate only TEM. Conversely, expressing a chimeric CCR2 ligand engineered to bind glycosaminoglycans on ECs results in CCR2-mediated arrest but blocks TEM induction. Our results from human pathogenic-like type 17 cells thus suggest that T cell migration arrest requires chemokine bound to EC surfaces, whereas TEM requires a transendothelial chemokine gradient.

Control of Mycobacterium tuberculosis (Mtb) infection requires generation of T cells that migrate to granulomas, complex immune structures surrounding sites of bacterial replication. Here we compared the gene expression profiles of T cells in pulmonary granulomas, bronchoalveolar lavage, and blood of Mtb-infected rhesus macaques to identify granuloma-enriched T cell genes. TNFRSF8/CD30 was among the top genes upregulated in both CD4 and CD8 T cells from granulomas. In mice, CD30 expression on CD4 T cells is required for survival of Mtb infection, and there is no major role for CD30 in protection by other cell types. Transcriptomic comparison of WT and CD30−/− CD4 T cells from the lungs of Mtb-infected mixed bone marrow chimeric mice showed that CD30 directly promotes CD4 T cell differentiation and the expression of multiple effector molecules. These results demonstrate that the CD30 co-stimulatory axis is highly upregulated on granuloma T cells and is critical for protective T cell responses against Mtb infection.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Roseomonas mucosa treatment in children with atopic dermatitis is associated with clinical improvements and potentially lipid-mediated TNF signaling.

PAX1deficiency causes a syndromic form of SCID by altering differentiation of thymic epithelial cells and other pharyngeal pouch tissues.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

Extracellular vesicles (EVs) mediate the intercellular transfer of RNAs, which alter gene expression in target cells. EV heterogeneity has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing Jurkat T cell EVs captured using CD47, CD63, and MHC1 antibodies. These EV subsets have similar sizes but divergent RNA contents. Apart from differences in numbers of nonannotated transcripts, CD63⁺, MHC1⁺, and CD47⁺EVs have similar overall contents of most noncoding RNA classes, but the relative enrichment of specific RNAs differs. The enrichment of micro-RNAs is highly divergent, and some including miR320a are selectively concentrated in CD47⁺EVs. Small nucleolar RNAs including SNORD116@ and SNHG10 are also selectively enriched in CD47⁺EVs, whereas no small nuclear RNAs are enriched in CD47⁺EVs. Conversely, MHC1⁺EVs are selectively enriched in a subset of tRNAs including TRE-CTC and TRR-CCG. This heterogeneity in RNA composition suggests multiple sorting mechanisms that direct specific RNAs into subsets of EVs that express specific surface markers.