The local microenvironment drives activation of neutrophils in human brain tumors.

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

Immunogenomic analysis of human brain metastases reveals diverse immune landscapes across genetically distinct tumors.

Brain metastases (BrMs) are the most common form of brain tumors in adults and frequently originate from lung and breast primary cancers. BrMs are associated with high mortality, emphasizing the need for more effective therapies. Genetic profiling of primary tumors is increasingly used as part of the effort to guide targeted therapies against BrMs, and immune-based strategies for the treatment of metastatic cancer are gaining momentum. However, the tumor immune microenvironment (TIME) of BrM is extremely heterogeneous, and whether specific genetic profiles are associated with distinct immune states remains unknown. Here, we perform an extensive characterization of the immunogenomic landscape of human BrMs by combining whole-exome/whole-genome sequencing, RNA sequencing of immune cell populations, flow cytometry, immunofluorescence staining, and tissue imaging analyses. This revealed unique TIME phenotypes in genetically distinct lung- and breast-BrMs, thereby enabling the development of personalized immunotherapies tailored by the genetic makeup of the tumors.

Influence of HLADQA1*05 Genotype in Adults With Inflammatory Bowel Disease and Anti-TNF Treatment With Proactive Therapeutic Drug Monitoring: A Retrospective Cohort Study.

BACKGROUND: Carriers of the human leucocyte antigen variant HLADQA1*05 (rs2097432) are at risk of developing antibodies against infliximab and adalimumab with reduced tumor necrosis factor (TNF) antagonist persistence. The impact of proactive therapeutic drug monitoring (PTDM) on this association has been barely assessed. METHODS: We conducted a retrospective single-center cohort study including patients with inflammatory bowel disease starting anti-TNF therapy between January 2017 and March 2021. Proactive therapeutic drug monitoring was defined as periodic drug level measurement (≥2 determinations during the first year of treatment and ≥1/annual determination during the following years), regardless of clinical condition, followed by dose optimization. Variables associated with treatment persistence were assessed with multivariable Cox regression analysis. RESULTS: A total of 112 patients were included, 52 (46.4%) HLA-DQA1*05 carriers, with a median follow-up of 73.9 (interquartile range, 35.4-133.1) weeks. Combination therapy with thiopurines was more frequent among HLA-DQA1*05 noncarriers (28 [46.7%] vs 12 [23.1%]; P = .01). Clinical remission rates at week 14 (77.9% vs 73.9%; P = .69) and 56 (73.2% vs 68.4%; P = .64) were similar between HLA-DQA1*05 noncarriers and carriers. Drug persistence was higher among HLA-DQA1*05 carriers (hazard ratio [HR], 0.32; 95% confidence interval, 0.14-0.71; P = .01). Multivariable Cox regression analysis identified systemic steroids at anti-TNF initiation (HR, 4; 95% confidence interval, 1.7-9.7) as a risk factor and HLA-DQA1*05 carriers (HR, 0.31; 95% confidence interval, 0.12-0.81) as a protective factor of treatment cessation. CONCLUSION: In adult patients with PTDM, a positive HLA-DQA1*05 genotype does not associate a higher risk of treatment cessation nor worse clinical outcomes.

This is a retrospective cohort study including 112 inflammatory bowel disease patients starting anti-TNF therapy under proactive therapeutic drug monitoring (PTDM). The HLA-DQA1*05 carriers did not present lower drug persistence or remission rates, suggesting PTDM overcomes the reduced treatment survival expected in HLA-DQA1*05 carriers.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.

Human intestinal dendritic cell and macrophage subsets in coeliac disease.

Dendritic cells (DC) and macrophages (Mϕ) constitute the most abundant antigen presenting cells in the human intestinal mucosa. In resting conditions, they are essential to maintain the mechanisms of immune tolerance toward food antigens and commensals, at the time that they keep the capacity to initiate and maintain antigen-specific pro-inflammatory immune responses toward invading pathogens. Nevertheless, this delicate equilibrium between immunity and tolerance is not perfect, like in coeliac disease (CD), where DC and Mϕ drive the development of antigen-specific immune responses toward dietary gluten peptides. In this review, we provide therefore a comprehensive discussion about CD pathogenesis, the human intestinal immune system and the biology of intestinal DC and Mϕ both in resting conditions and in CD. Last, but not least, we discuss about all the remaining issues pending to be studied regarding DC and Mϕ contribution toward CD pathogenesis. This may allow the identification of unique and specific factors which may be useful in the clinical practice, as well as identify new therapeutic targets in order to reestablish the loss intestinal homeostasis in CD.

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.

Frequent mutations in the amino-terminal domain of BCL7A impair its tumor suppressor role in DLBCL.

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.

Infectious stimuli promote malignant B-cell acute lymphoblastic leukemia in the absence of AID.

The prerequisite to prevent childhood B-cell acute lymphoblastic leukemia (B-ALL) is to decipher its etiology. The current model suggests that infection triggers B-ALL development through induction of activation-induced cytidine deaminase (AID; also known as AICDA) in precursor B-cells. This evidence has been largely acquired through the use of ex vivo functional studies. However, whether this mechanism governs native non-transplant B-ALL development is unknown. Here we show that, surprisingly, AID genetic deletion does not affect B-ALL development in Pax5-haploinsufficient mice prone to B-ALL upon natural infection exposure. We next test the effect of premature AID expression from earliest pro-B-cell stages in B-cell transformation. The generation of AID off-target mutagenic activity in precursor B-cells does not promote B-ALL. Likewise, known drivers of human B-ALL are not preferentially targeted by AID. Overall these results suggest that infections promote B-ALL through AID-independent mechanisms, providing evidence for a new model of childhood B-ALL development.

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation.

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation.

miR-28 regulates the germinal center reaction and blocks tumor growth in preclinical models of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma comprises a variety of neoplasms, many of which arise from germinal center (GC)-experienced B cells. microRNA-28 (miR-28) is a GC-specific miRNA whose expression is lost in numerous mature B-cell neoplasms. Here we show that miR-28 regulates the GC reaction in primary B cells by impairing class switch recombination and memory B and plasma cell differentiation. Deep quantitative proteomics combined with transcriptome analysis identified miR-28 targets involved in cell-cycle and B-cell receptor signaling. Accordingly, we found that miR-28 expression diminished proliferation in primary and lymphoma cells in vitro. Importantly, miR-28 reexpression in human Burkitt (BL) and diffuse large B-cell lymphoma (DLBCL) xenografts blocked tumor growth, both when delivered in viral vectors or as synthetic, clinically amenable, molecules. Further, the antitumoral effect of miR-28 is conserved in a primary murine in vivo model of BL. Thus, miR-28 replacement is uncovered as a novel therapeutic strategy for DLBCL and BL treatment.

Self-assembled protein arrays from an Ornithodoros moubata salivary gland expression library.

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

Feocromocitoma extraadrenal: actualización de los aspectos más controvertidos. A propósito de dos casos / Extra-adrenal pheochromocytoma: update of the most controversial features. A propos of two cases

Los feocromocitomas son tumores neuroendocrinospoco frecuentes, habitualmente sintomáticos y en la mayoría de los casos esporádicos, que se suelen localizar en la médula suprarrenal. Presentamos 2 casos de feocromocitoma extraadrenal no funcionan-te localizados en el retroperitoneo, aparentemente benignos, esporádicos y no asociados con otras neoplasias endocrinas ni enfermedades hereditarias. Empleamos ecografía, tomografía computarizada y resonancia magnética antes de la escisión completa del tumor por vía abdominal abierta, así como diversas técnicas inmunohistoquímicas para llegar al diagnóstico definitivo. Tras 8 y 14 meses de seguimiento no hay evidencia de recidiva de la enfermedad ni de aparición de otros síndromes. Como consecuencia de estos casos, llevamos a cabo una revisión bibliográfica centrándonos en los puntos más controvertidos (AU)

Pheochromocytomas are uncommon neuroendocrine tumors. They are usually symptomatic and sporadic and are generally located in the adrenal medulla. We present two cases of extra-adrenal nonfunctional pheochromocytomas located in the retro peritoneum. The tumors were apparently benign and sporadic and were not associated with other neoplasms or hereditary diseases. Ultrasonography, computed axial tomography and magnetic resonance imaging were performed before complete excision of the tumors using open abdominal surgery. Several immunohistochemical techniques were performed to reach the definitive diagnosis. After 8 and 14 months of follow-up, there is no evidence of recurrence or other syndromes. As a consequence of these two cases, we performed review of the literature on the topic, focussing on the most controversial areas (AU)

[Extra-adrenal pheochromocytoma: update of the most controversial features. Apropos of two cases]. / Feocromocitoma extraadrenal: actualización de los aspectos más controvertidos. A propósito de dos casos.

Pheochromocytomas are uncommon neuroendocrine tumors. They are usually symptomatic and sporadic and are generally located in the adrenal medulla. We present two cases of extra-adrenal nonfunctional pheochromocytomas located in the retroperitoneum. The tumors were apparently benign and sporadic and were not associated with other neoplasms or hereditary diseases. Ultrasonography, computed axial tomography and magnetic resonance imaging were performed before complete excision of the tumors using open abdominal surgery. Several immunohistochemical techniques were performed to reach the definitive diagnosis. After 8 and 14 months of follow-up, there is no evidence of recurrence or other syndromes. As a consequence of these two cases, we performed a review of the literature on the topic, focussing on the most controversial areas.

Efectividad del piroxicam en supositorios y del diclofenac (tabletas y supositorios) en pacientes con gonartrosis / Effectivity of piroxicam in suppositories and diclofenac (tablets and suppositories) in patients with gonarthrosis

Se seleccionaron 43 pacientes de ambos sexos con una edad comprendida entre 46 a 80 años, en cuatro Servicios de Reumatología integrados al Centro Nacional de Enfermedades Reumáticas del MSAS. Los pacientes presentaron inflamación, dolor en reposo, dolor a la presión, dolor a la movilización activa y pasiva de la articulación de una o ambas rodillas y limitación de la actividad física diaria. Todas las personas incluidas dieron su consentimiento para participar en este estudio. Excluyendo aquellos con úlcera péptica, embarazadas, madres amamantando y alérgicos a la aspirina u otros antiinflamatorios no esteroides (AINES). Los pacientes fueron tratados por 15 días con 20 mg de piroxicam en supositorios, y luego de una semana sin droga se comenzó la administración por 15 días más de diclofenac a la dosis diaria de 50 mg por vía oral. El dolor en reposo disminuyó en el 35.7% de los pacientes tratados con piroxicam en comparación con 8.4% por diclofenac. Hubo mejoría de la movilización pasiva de la rodilla con ambas drogas, sin diferencias significativas entre ellas. El dolor a la presión en la cara interna de la rodilla disminuyó significativamente más con el piroxicam que con el diclofenac. El dolor a la presión en la cara externa de la rodilla mejoró con ambos tratamientos, pero no hubo diferencia significativa entre los dos tipos de medicamento, igualmente ocurrió con el dolor al caminar y la actividad diaria del paciente. La velocidad para caminar 20 metros fue mejorada con los dos tratamientos en un rango de 0.1 a 2.5 segundos, sin embargo esta mejoría no fue estadísticamente significativa..