SARS-CoV-2-specific serological and functional T cell immune responses during acute and early COVID-19 convalescence in solid organ transplant patients.

The description of protective humoral and T cell immune responses specific against SARS-CoV-2 has been reported among immunocompetent (IC) individuals developing COVID-19 infection. However, its characterization and determinants of poorer outcomes among the at-risk solid organ transplant (SOT) patient population have not been thoroughly investigated. Cytokine-producing T cell responses, such as IFN-γ, IL-2, IFN-γ/IL-2, IL-6, IL-21, and IL-5, against main immunogenic SARS-CoV-2 antigens and IgM/IgG serological immunity were tracked in SOT (n = 28) during acute infection and at two consecutive time points over the following 40 days of convalescence and were compared to matched IC (n = 16) patients admitted with similar moderate/severe COVID-19. We describe the development of a robust serological and functional T cell immune responses against SARS-CoV-2 among SOT patients, similar to IC patients during early convalescence. However, at the infection onset, SOT displayed lower IgG seroconversion rates (77% vs. 100%; p = .044), despite no differences on IgG titers, and a trend toward decreased SARS-CoV-2-reactive T cell frequencies, especially against the membrane protein (7 [0-34] vs. 113 [15-245], p = .011, 2 [0-9] vs. 45 [5-74], p = .009, and 0 [0-2] vs. 13 [1-24], p = .020, IFN-γ, IL-2, and IFN-γ/IL-2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID-19.

Telomere attrition in heart failure: a flow-FISH longitudinal analysis of circulating monocytes.

BACKGROUND: Cross-sectional investigations report shorter telomeres in patients with heart failure (HF); however, no studies describe telomere length (TL) trajectory and its relationship with HF progression. Here we aimed to investigate telomere shortening over time and its relationship to outcomes. METHODS: Our study cohort included 101 ambulatory patients with HF. Blood samples were collected at baseline (n = 101) and at the 1-year follow-up (n = 54). Using flow-FISH analysis of circulating monocytes, we simultaneously measured three monocyte subsets-classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++)-and their respective TLs based on FITC-labeled PNA probe hybridization. The primary endpoints were all-cause death and the composite of all-cause death or HF-related hospitalization, assessed at 2.3 ± 0.6 years. All statistical analyses were executed by using the SPSS 15.0 software, and included Student's t test and ANOVA with post hoc Scheffe analysis, Pearson or Spearman rho correlation and univariate Cox regression when applicable. RESULTS: We found high correlations between TL values of different monocyte subsets: CD14++CD16+ vs. CD14++CD16-, R = 0.95, p < 0.001; CD14++CD16+ vs. CD14+CD16++, R = 0.90, p < 0.001; and CD14++CD16- vs. CD14+CD16++, R = 0.89, p < 0.001. Mean monocyte TL exhibited significant attrition from baseline to the 1-year follow-up (11.1 ± 3.3 vs. 8.3 ± 2.1, p < 0.001). TL did not significantly differ between monocyte subsets at either sampling time-point (all p values > 0.1). Cox regression analyses did not indicate that TL or ΔTL was associated with all-cause death or the composite endpoint. CONCLUSIONS: Overall, this longitudinal study demonstrated a ~ 22% reduction of TL in monocytes from ambulatory patients with HF within 1 year. TL and ΔTL were not related to outcomes over long-term follow-up.

Brilliant violet fluorochromes in simultaneous multicolor flow cytometry-fluorescence in situ hybridization measurement of monocyte subsets and telomere length in heart failure.

Conventional analytical methods to determine telomere length (TL) have been replaced by more precise and reproducible procedures, such as fluorescence in situ hybridization coupled with flow cytometry (flow-FISH). However, simultaneous measurement of TL and cell phenotype remains difficult. Relatively expensive and time-consuming cell-sorting purification is needed to counteract the loss, due to stringent FISH conditions, of prehybridization fluorescence by the organic fluorochromes conventionally used in the phenotyping step. Here, we sought to assess whether the newly developed Brilliant Violet (BV) dyes are valuable to specifically and simultaneously assess the distribution and telomere attrition of monocyte subsets circulating in the blood of a cohort of patients with heart failure. We performed flow-FISH on blood samples from 28 patients with heart failure. To differentiate among monocyte subsets, we used BV and conventional fluorochromes conjugated to antibodies against CD86, CD14, CD16, and CD15. We simultaneously assessed the TLs of the monocyte subsets with a telomere-specific peptide nucleic acid probe labeled with fluorescein isothiocyanate. The BV dyes completely tolerated the harsh conditions required for adequate DNA denaturation and simultaneously provided accurate identification of monocyte subpopulations and respective TLs. The presented protocol may be faster and less expensive than those used currently for purposes such as establishing associations among patient categories, disease progression, monocyte heterogeneity, and aging in the context of heart failure.

Cyclophosphamide-free mobilisation increases safety while preserving the efficacy of autologous haematopoietic stem cell transplantation in refractory Crohn's disease patients.

BACKGROUND AND AIM: Autologous haematopoietic stem cell transplantation [AHSCT] is a therapeutic option for refractory Crohn's disease [CD]. However, high adverse event rates related to chemotherapy toxicity and immunosuppression limit its applicability. This study aims to evaluate AHSCT's safety and efficacy using a cyclophosphamide (Cy)-free mobilisation regimen. METHODS: A prospective observational study included 14 refractory CD patients undergoing AHSCT between June 2017 and October 2022. The protocol involved outpatient mobilisation with G-CSF 12-16 µg/kg/daily for 5 days, and optional Plerixafor 240 µg/d (1-2 doses) if the CD34+ cell count target was unmet. Standard conditioning with Cy and anti-thymocyte globulin was administered. Clinical, endoscopic, and radiological assessments were conducted at baseline and during follow-up. RESULTS: All patients achieved successful outpatient mobilisation (7 patients needed Plerixafor) and underwent transplantation. Median follow-up was 106 weeks (IQR 52-348). No mobilisation-related serious adverse events (SAEs) or CD worsening occurred. Clinical and endoscopic remission rates were 71% and 41.7% at 26 weeks, 64% and 25% at 52 weeks, and 71% and 16.7% at the last follow-up. The percentage of patients who restarted CD therapy for clinical relapse and/or endoscopic/radiological activity was 14% at 26 weeks, 57% at 52 weeks, and 86% at the last follow-up. Peripheral blood cell populations and antibody levels post-AHSCT were comparable to Cy-based mobilisation. CONCLUSIONS: Cy-free mobilisation is safe and feasible in refractory CD patients undergoing AHSCT. Although relapse occurs in a significant proportion of patients, clinical and endoscopic responses are achieved upon CD-specific therapy reintroduction.

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease.

Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity.

Circulating monocyte subsets and heart failure prognosis.

Monocytes are a heterogeneous population of effector cells with key roles in tissue integrity restoration and maintenance. Here, we explore the association of monocyte subsets and prognosis in patients with ambulatory heart failure (HF). Monocyte subsets were classified as classical (CD14++/CD16-), intermediate (CD14++/CD16+), or non-classical (CD14+/CD16++). Percentage distribution and absolute cell count were assessed in each subset, and multivariable Cox regression analyses were performed with all-cause death, HF-related hospitalization, and the composite end-point of both as dependent variables. 400 patients were consecutively included (72.8% male, age 69.4±12.2 years, 45.5% from ischemic aetiology, left ventricle ejection fraction (LVEF) 41.6% ±14.5, New York Heart Association (NYHA) class II 62.8% and III 30.8%). During a mean follow-up of 2.6±0.9 years, 107 patients died, 99 had a HF-related hospitalization and 160 suffered the composite end-point of all-cause death or HF-related hospitalization. Monocyte subsets assessed in percentages were not independently associated to any of the end-points. When considering number of cells/µL, intermediate subset was independently associated with an increase of all-cause death (HR 1.25 [95% CI 1,02-1.52], p = 0.03), and the composite end-point HR 1.20 [95% CI 1,03-1.40], p = 0.02). The presented findings show that absolute cell count of monocyte subsets was preferred over monocyte percentage for prognosis stratification for outpatients with HF. The intermediate monocyte subset provides information on increased risk of all-cause death and the composite end-point.