Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.

Lineage tracing reveals B cell antibody class switching is stochastic, cell-autonomous, and tuneable.

To optimize immunity to pathogens, B lymphocytes generate plasma cells with functionally diverse antibody isotypes. By lineage tracing single cells within differentiating B cell clones, we identified the heritability of discrete fate controlling mechanisms to inform a general mathematical model of B cell fate regulation. Founder cells highly influenced clonal plasma-cell fate, whereas class switch recombination (CSR) was variegated within clones. In turn, these CSR patterns resulted from independent all-or-none expression of both activation-induced cytidine deaminase (AID) and IgH germline transcription (GLT), with the latter being randomly re-expressed after each cell division. A stochastic model premised on these molecular transition rules accurately predicted antibody switching outcomes under varied conditions in vitro and during an immune response in vivo. Thus, the generation of functionally diverse antibody types follows rules of autonomous cellular programming that can be adapted and modeled for the rational control of antibody classes for potential therapeutic benefit.

Survey of activation-induced genome architecture reveals a novel enhancer of Myc.

The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of Myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 h post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

CXCL11 expressing C57BL/6 mice have intact adaptive immune responses to viral infection.

The chemokine receptor CXCR3 is expressed on immune cells to co-ordinate lymphocyte activation and migration. CXCR3 binds three chemokine ligands, CXCL9, CXCL10 and CXCL11. These ligands display distinct expression patterns and ligand signaling biases; however, how each ligand functions individually and collaboratively is incompletely understood. CXCL9 and CXCL10 are considered pro-inflammatory chemokines during viral infection, while CXCL11 may induce a tolerizing state. The investigation of the individual role of CXCL11 in vivo has been hampered as C57BL/6 mice carry several mutations that result in a null allele. Here, CRISPR/Cas9 was used to correct these mutations on a C57BL/6 background. It was validated that CXCL11KI mice expressed CXCL11 protein in dendritic cells, spleen and lung. CXCL11KI mice were largely phenotypically indistinguishable from C57BL/6 mice, both at steady-state and during two models of viral infection. While CXCL11 expression did not modify acute antiviral responses, this study provides a new tool to understand the role of CXCL11 in other experimental settings.

[Adaptation and validation of checklists to detect medication prescription and dispensing errors]. / Adaptación y validación de instrumentos para medir errores en las recetas médicas y en el proceso de preparación de medicamentos en farmacia de pacientes ambulatorios.

BACKGROUND: Adverse effects of medications are an important source of morbidity. Prescription and dispensing errors are an important cause of these adverse effects. AIM: To adapt and validate two checklists, one to measure errors in handwritten prescriptions and other to detected errors in the medication dispensing process of hospital pharmacies for outpatient care. MATERIAL AND METHODS: The study was conducted in three stages. First, checklists for medication errors developed elsewhere were adapted. Afterwards, the checklists were reviewed by experts. Finally, the inter and intra-observer reliability of each checklist was assessed, testing them in 32 occasions by two independent observers. RESULTS: The checklists for medication prescription and dispensing were composed by 12 and seven items, respectively. They were corrected according to experts' opinions. The intraclass correlations of the results of each tester were 0.68 and 0.82 for the prescription and dispensing error checklists, respectively. CONCLUSIONS: The developed checklists for the detection of errors in prescription and dispensing of medications are reliable en can be applied in future studies.

Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles.

Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (BRM) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza-specific BRM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)- and nucleoprotein (NP)-specific lung BRM. We found that CCR6 facilitates increased recruitment and/or retention of BRM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning BRM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that BRM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.

Synthesis of aminooxy and N-alkylaminooxy amines for use in bioconjugation.

Five Boc-protected aminooxy and N-alkylaminooxy amines have been synthesized in 60-95% overall yield using a common synthetic strategy from readily available two- and three-carbon Cbz-protected amino alcohols. The amines can be linked to biomolecules via amide formation and incorporated directly into peptoids via submonomer synthesis. Subsequent deprotection of the aminooxy and N-alkylaminooxy groups enables conjugation with desired target molecules via established chemoselective ligation methods. The range of derivatives synthesized allows different distances to be established between the conjugated molecules.

PREVALENCE OF COLUMBID HERPESVIRUS INFECTION IN FERAL PIGEONS FROM NEW SOUTH WALES AND VICTORIA, AUSTRALIA, WITH SPILLOVER INTO A WILD POWERFUL OWL (NINOX STRUENA).

Columbid herpesvirus-1 (CoHV-1) is widespread in feral pigeons in North America and Europe. We used a PCR assay to detect CoHV-1 DNA in oral and cloacal tissues and oral swabs from naturally infected pigeons. Fifty-three feral pigeons from five flocks in Australia (n=3 from south-central Victoria and n=2 from Sydney) were examined for CoHV-1 DNA. We detected CoHV-1 DNA in oral mucosa and cloacal mucosa, with higher concentrations in the oral mucosa. The sensitivity of testing oral swabs was the same as testing the tissue, indicating that testing of oral swabs from live birds is an effective means of screening flocks for CoHV-1 infection. Infection was found in all five of the flocks examined and the prevalence of infection ranged from 70% to100%. Most positive birds could be detected with a single-amplification PCR, but a nested amplification was required to detect others. Oral swabs from Australian native doves and pigeons (n=18) and the introduced Collared Dove (Streptopelia chinensis; n=2) were also tested by the nested PCR and all were negative for CoHV-1 DNA. We describe a fatal infection of CoHV-1 in a wild Powerful Owl (Ninox strenua) that was observed feeding on feral pigeons. This is the first known case of CoHV-1 causing death in a wild bird of prey in Australia. Our data suggest that CoHV-1 is widespread in feral pigeon flocks in Australia but we did not find it in native doves and pigeons. Spillover into native avian predator species may be occurring.

Impacto clínico de un grupo de discusión multidisciplinaria en el diagnóstico de fibrosis pulmonar idiopática en Colombia / Clinical impact of a multidisciplinary discussion group on the diagnosis of idiopathic pulmonary fibrosis in Colombia

Resumen Introducción: la fibrosis pulmonar idiopática (FPI) es una enfermedad pulmonar intersticial (EPID) de mal pronóstico, considerada huérfana en Colombia. Un diagnóstico correcto tiene implicaciones para el paciente y los costos de atención. Los grupos de discusión multidisciplinaria (GDM) se consideran el estándar de oro en el diagnóstico. No hay estudios previos en Colombia de la experiencia de un GDM. Objetivos: evaluar el impacto de un GDM en una institución de cuarto nivel en Bogotá en cambio de diagnóstico de pacientes con EPID y la concordancia entre el diagnóstico inicial y final de FPI. Material y métodos: pacientes con EPID evaluados entre 2015-2018 por el GDM conformado por neumólogos, radiólogo, patólogo y reumatólogos. Criterios ATS/ERS/JRS/ALAT de diagnóstico de FPI. Descripción del cambio en el diagnóstico y concordancia entre el diagnóstico inicial y del GDM en FPI. Resultados: de 165 pacientes con EPID se cambió el diagnóstico en 35.2%. En 77.3% pacientes con diagnóstico inicial de FPI y en 6.7% con diagnóstico inicial diferente a FPI el GDM confirmó FPI. Al descartar FPI, los principales diagnósticos fueron neumonitis de hipersensibilidad en fase crónica (29.4%) y neumonía intersticial no específica (23.5%). El índice kappa entre el diagnóstico inicial y final de FPI fue 0.71 (0.60-0.82). Conclusiones: el GDM en EPID tuvo un importante impacto clínico demostrado por un alto porcentaje de cambió del diagnóstico de remisión. Se descartó el diagnóstico inicial de FPI en un porcentaje significativo de pacientes y se ratificó en un grupo menor sin esta sospecha clínica inicial. (Acta Med Colomb 2022; 47. DOI:https://doi.org/10.36104/amc.2022.2017).

Abstract Introduction: idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) with a poor prognosis, considered an orphan disease in Colombia. An accurate diagnosis has implications for the patient and healthcare costs. Multidisciplinary discussion groups (MDGs) are considered the gold standard for diagnosis. There are no prior studies in Colombia on the experience of an MDG. Objectives: to evaluate the impact of an MDG in a quaternary care institution in Bogotá on the change in the diagnosis of patients with ILD and the concordance between the initial and final diagnosis of IPF. Materials and methods: patents with ILD evaluated from 2015-2018 by the MDG made up of pulmonologists, a radiologist, a pathologist and rheumatologists. The ATS/ERS/JRS/ALAT diagnostic criteria for IPF. A description of changes in the diagnosis and the agreement between the initial diagnosis and the MDG diagnosis of IPF. Results: out of 165 patients with ILD, the diagnosis was changed in 32.5%. The MDG confirmed IPF in 77.3% of patients with an initial diagnosis of ILD and 6.7% of those with a different initial diagnosis. When IPF was ruled out, the main diagnoses were chronic hypersensitivity pneumonitis (24.8%) and nonspecific interstitial pneumonia (23.5%). The Kappa index between the initial and final IPF diagnoses was 0.71 (0.60-0.82). Conclusions: the MDG on ILD had a significant clinical impact evidenced by a high percentage of change in the referral diagnosis. The initial diagnosis of IPF was ruled out in a significant percentage of patients and confirmed in a smaller group which did not have this initial clinical suspicion. (Acta Med Colomb 2022; 47. DOI:https://doi.org/10.36104/amc.2022.2017).

[The lymph nodes imprint for the diagnosis of lymphoid neoplasms]. / La impronta del ganglio linfático para el diagnóstico de neoplasias linfoides.

BACKGROUND: lymphoma is the most frequent lymphoid neoplasm in our country. Its diagnosis is based on histopathological findings. The lymph node imprint has been used for more than 40 years. The aim was to establish the sensitivity, specificity, positive predictive value and negative predictive value of lymph node imprint and estimate the inter-observer rate. METHODS: we did an observational, retrospective, prolective study, based on the lymph node imprint obtained by excisional biopsies over a period of 6 years. RESULTS: the inclusion criteria was met on 199 samples, 27.1 % were considered as reactive (n = 54), 16.1 % Hodgkin lymphoma (n = 32), 40.2 % (n = 80) non-Hodgkin lymphoma and 16.6 % (n = 33) as metastatic carcinoma. Comparing with the final histopathology report, the sensitivity and specificity of lymph node imprint were 88 % (0.81-0.95) and 64 % (0.55-0.73) respectively, the positive predictive value was 67 % (0.59-0.76) and the negative predictive value was 86 % (0.79-0.94). The interobserver kappa index was 0.467. CONCLUSIONS: the lymph node imprint remains as a useful tool for the diagnosis of lymphoid neoplasm. The agreement between observers was acceptable.

Introducción: los linfomas son las neoplasias linfoproliferativas más frecuentes en México. Su diagnóstico se basa en el estudio histopatológico. El análisis morfológico de la impronta del ganglio linfático se ha utilizado desde hace más de 40 años. Los objetivos de esta investigación fueron determinar la sensibilidad, la especificidad y los valores predictivos positivo y negativo de la impronta del ganglio linfático para el diagnóstico del linfoma y estimar el índice interobservador. Métodos: estudio retrospectivo, observacional y prolectivo de las improntas de ganglios linfáticos obtenidas por biopsia durante un periodo de seis años. Resultados: se incluyeron 199 improntas; 27.1 % se consideró reactivo (n = 54), 16.1 % como linfoma Hodgkin (n = 32), 40.2 % (n = 80) como no Hodgkin y 16.6 % (n = 33) como carcinoma metastásico. Al compararlas con los reportes histopatológicos finales, la sensibilidad fue de 88 % (0.81-0.95), la especificidad de 64 % (0.55-0.73), el valor predictivo positivo de 67 % (0.59-0.76) y el negativo, de 86 % (0.79-0.94). La concordancia interobservador fue moderada (índice kappa de 0.467). Conclusiones: el análisis de la impronta del ganglio linfático es útil para el diagnóstico de las neoplasias linfoproliferativas.