Regulation of Foxo1 expression is critical for central B cell tolerance and allelic exclusion.

Resolving the molecular mechanisms of central B cell tolerance might unveil strategies that prevent autoimmunity. Here, using a mouse model of central B cell tolerance in which Forkhead box protein O1 (Foxo1) is either deleted or over-expressed in B cells, we show that deleting Foxo1 blocks receptor editing, curtails clonal deletion, and decreases CXCR4 expression, allowing high-avidity autoreactive B cells to emigrate to the periphery whereby they mature but remain anergic and short lived. Conversely, expression of degradation-resistant Foxo1 promotes receptor editing in the absence of self-antigen but leads to allelic inclusion. Foxo1 over-expression also restores tolerance in autoreactive B cells harboring active PI3K, revealing opposing roles of Foxo1 and PI3K in B cell selection. Overall, we show that the transcription factor Foxo1 is a major gatekeeper of central B cell tolerance and that PI3K drives positive selection of immature B cells and establishes allelic exclusion by suppressing Foxo1.

Pharmacogenomic insights in psychiatric care: uncovering novel actionability, allele-specific CYP2D6 copy number variation, and phenoconversion in 15,000 patients.

Pharmacogenomic testing has emerged as an aid in clinical decision making for psychiatric providers, but more data is needed regarding its utility in clinical practice and potential impact on patient care. In this cross-sectional study, we determined the real-world prevalence of pharmacogenomic actionability in patients receiving psychiatric care. Potential actionability was based on the prevalence of CYP2C19 and CYP2D6 phenotypes, including CYP2D6 allele-specific copy number variations (CNVs). Combined actionability additionally incorporated CYP2D6 phenoconversion and the novel CYP2C-TG haplotype in patients with available medication data. Across 15,000 patients receiving clinical pharmacogenomic testing, 65% had potentially actionable CYP2D6 and CYP2C19 phenotypes, and phenotype assignment was impacted by CYP2D6 allele-specific CNVs in 2% of all patients. Of 4114 patients with medication data, 42% had CYP2D6 phenoconversion from drug interactions and 20% carried a novel CYP2C haplotype potentially altering actionability. A total of 87% had some form of potential actionability from genetic findings and/or phenoconversion. Genetic variation detected via next-generation sequencing led to phenotype reassignment in 22% of individuals overall (2% in CYP2D6 and 20% in CYP2C19). Ultimately, pharmacogenomic testing using next-generation sequencing identified potential actionability in most patients receiving psychiatric care. Early pharmacogenomic testing may provide actionable insights to aid clinicians in drug prescribing to optimize psychiatric care.

Autoreactive Antibodies Associated with Castleman Disease Triad.

The Castleman triad has been described in a select few patients presenting with a retroperitoneal mass, mucocutaneous pemphigus vulgaris, and bronchiolitis obliterans. Here, we describe the Castleman triad in a 19-year-old male with unicentric hyaline vascular type Castleman disease (HV-CD). This patient presented with an array of positive antibodies, including anti-cyclic citrullinated peptide, anti-double-stranded DNA, and Sjogren's IgG. Interestingly, the patient's rheumatologic symptoms resolved after tumor resection, while his antibody profile remained relatively unchanged. HV-CD, with a triad presentation, was thought to be from a paraneoplastic syndrome secondary to an underlying lymphoproliferative disorder. The findings presented here identify multiple autoantibodies potentially contributing to this patient's presentation with HV-CD.

Autologous platelet-rich plasma (APRP) in diabetes foot disease: a meta-analysis.

INTRODUCTION: This study will explore the effectiveness of autologous platelet-rich plasma in the treatment of diabetic foot disease compared to conventional treatments, based on the ulcer healing rate. METHODS: The electronic databases of PubMed, EMBASE, and WOS internet were searched. Evaluated outcome rate of complete ulcer healing. Statistical analysis was performed with RevMan 5.0 software and SPSS 25.0. RESULTS: Eleven RCTs with 828 patients were included in this study. The meta-analysis showed a higher complete ulcer healing rate (OR = 3.69, 95 % CI 2.62 to 5.20, P < 0.01, I2 = 0 %) in growth factors based in autologous platelech-rich plasma (aPRP) group compared with control. Mixed evidence was seen for publication bias, but analyses by using the trim-and-fill method did not appreciably alter results. CONCLUSION: Autologous platelet-rich plasma can improve the complete healing rate of the ulcer compared to current conventional treatments in diabetic foot ulcer patients.

Improving spin crossover characteristics in heteroleptic [FeIII(qsal-5-I)(qsal-5-OMe)]A complexes.

A family of heteroleptic spin crossover (SCO) [FeIII(qsal-5-I)(qsal-5-OMe)]A·sol (qsal-5-X = 5-X-2-[(8-quinolylimino)methyl]phenolate; A = NO3-1 sol = 2MeOH, NCS-2 sol = 0.75MeOH·1.3H2O, BF4-3 sol = MeOH, OTf-4, sol = MeOH) complexes have been synthesized. Most of the complexes exhibit gradual SCO, with the exception of NCS, which is principally high spin. In contrast, the OTf complex shows an abrupt hysteretic SCO (35 K) after solvent loss. The magnetic properties of this complex are significantly improved in comparison to the related homoleptics, [Fe(qsal-I)2]OTf 5 (hysteresis, 8 K) and [Fe(qsal-5-OMe)2]OTf·CH2Cl26 (gradual SCO). Structural studies reveal that slight changes in the crystal packing cause stronger interactions improving the cooperativity. These findings are supported by DFT calculations using the r2SCAN functional in which the calculated structures show that SCO from the LS to the HS state causes pronounced scissoring of the 1D π-π chains and substantial changes in their relative orientation following loss of MeOH.

Cartilage Defect Treatment Using High-Density Autologous Chondrocyte Implantation (HD-ACI).

Hyaline cartilage's inability to self-repair can lead to osteoarthritis and joint replacement. Various treatments, including cell therapy, have been developed for cartilage damage. Autologous chondrocyte implantation (ACI) is considered the best option for focal chondral lesions. In this article, we aimed to create a narrative review that highlights the evolution and enhancement of our chondrocyte implantation technique: High-Density-ACI (HD-ACI) Membrane-assisted Autologous Chondrocyte Implantation (MACI) improved ACI using a collagen membrane as a carrier. However, low cell density in MACI resulted in softer regenerated tissue. HD-ACI was developed to improve MACI, implanting 5 million chondrocytes per cm2, providing higher cell density. In animal models, HD-ACI formed hyaline-like cartilage, while other treatments led to fibrocartilage. HD-ACI was further evaluated in patients with knee or ankle defects and expanded to treat hip lesions and bilateral defects. HD-ACI offers a potential solution for cartilage defects, improving outcomes in regenerative medicine and cell therapy. HD-ACI, with its higher cell density, shows promise for treating chondral defects and advancing cartilage repair in regenerative medicine and cell therapy.

Activation and pro-inflammatory cytokine production by unswitched memory B cells during SARS-CoV-2 infection.

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells are debated in the literature, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells have been described to be reduced in frequency with severe SARS-CoV2 infection and here we characterize their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those who have had mild and severe SARS-CoV2 infection, confirm a reduction in the frequency of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this reduction is associated with increased levels of systemic TNFα. We further document how severe viral infection is associated with an increased frequency of 'IgD+' only memory B cells that correlate with increased IgG autoantibody levels. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with a concomitant reduction in the expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling-competent BCR that displayed enhanced BCR signaling activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection are poised to secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data indicate that B cells contribute to the inflammatory milieu during viral infection.

Bidirectional photoswitchability in an iron(iii) spin crossover complex: symmetry-breaking and solvent effects.

The impact of solvent on spin crossover (SCO) behaviour is reported in two solvates [Fe(qsal-I)2]NO3·2ROH (qsal-I = 4-iodo-2-[(8-quinolylimino)methyl]phenolate; R = Me 1 or Et 2) which undergo abrupt and gradual SCO, respectively. A symmetry-breaking phase transition due to spin-state ordering from a [HS] to [HS-LS] state occurs at 210 K in 1, while T1/2 = 250 K for the EtOH solvate, where complete SCO occurs. The MeOH solvate exhibits LIESST and reverse-LIESST from the [HS-LS] state, revealing a hidden [LS] state. Moreover, photocrystallographic studies on 1 at 10 K reveal re-entrant photoinduced phase transitions to a high symmetry [HS] phase when irradiated at 980 nm or a high symmetry [LS] phase after irradiation at 660 nm. This study represents the first example of bidirectional photoswitchability and subsequent symmetry-breaking from a [HS-LS] state in an iron(iii) SCO material.

Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity.

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity.

Regulation of CD8 T-cell signaling, metabolism, and cytotoxic activity by extracellular lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA2 , LPA5, and LPA6 , and work from our group has determined that LPA5 signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA5 axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA5 signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA5 inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA5 antagonism for the treatment of malignancies and chronic infections.

p27Kip1 V109G as a biomarker for CDK4/6 inhibitors indication in hormone receptor-positive breast cancer.

CDK4/6 inhibitors benefit a minority of patients who receive them in the breast cancer adjuvant setting. p27Kip1 is a protein that inhibits CDK/Cyclin complexes. We hypothesized that single-nucleotide polymorphisms that impaired p27Kip1 function could render patients refractory to endocrine therapy but responsive to CDK4/6 inhibitors, narrowing the patient subpopulation that requires CDK4/6 inhibitors. We found that the p27Kip1 V109G single-nucleotide polymorphism is homozygous in approximately 15% of hormone-positive breast cancer patients. Polymorphic patients experience rapid failure in response to endocrine monotherapy compared with wild-type or heterozygous patients in the first-line metastatic setting (progression-free survival: 92 vs 485 days, P < .001); when CDK4/6 inhibitors are added, the differences disappear (progression-free survival: 658 vs 761 days, P = .92). As opposed to wild-type p27Kip1, p27Kip1 V109G is unable to suppress the kinase activity of CDK4 in the presence of endocrine inhibitors; however, palbociclib blocks CDK4 kinase activity regardless of the p27Kip1 status. p27Kip1 genotyping could constitute a tool for treatment selection.

Crystal structure of a new europium(III) compound based on thio-phene-acrylic acid.

A europium(III) coordination compound based on thio-phene-acrylic acid (Htpa), tri-aqua-tris-[3-(thio-phen-2-yl)prop-2-enoato-κ2 O,O']europium(III)-3-(thio-phen-2-yl)prop-2-enoic acid (1/3), [Eu(C7H5O2S)3(H2O)3]·3C7H6O2S or [Eu(tpa)3(H2O)3]·3(Htpa) (1), where tpa is the conjugate base of Htpa, has been synthesized and structurally characterized. Compound 1 crystallizes in the trigonal space group R3. The structure of 1 consists of a discrete mol-ecular complex [Eu(tpa)3(H2O)3] species and the Htpa mol-ecule. In the crystal, the two components are involved in O-H⋯O [ring motif R 2 2(8)] and C-H⋯π hydrogen-bonding inter-actions. These inter-actions were further investigated by Hirshfeld surface analysis, which showed high contributions of H⋯H, H⋯C/C⋯H and H⋯O/O⋯H contacts to the total Hirshfeld surfaces.

CRISPR/Cas9-mediated gene editing. A promising strategy in hematological disorders.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the gene editing field, making it possible to interrupt, insert or replace a sequence of interest with high precision in the human genome. Its easy design and wide applicability open up a variety of therapeutic alternatives for the treatment of genetic diseases. Indeed, very promising approaches for the correction of hematological disorders have been developed in the recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. This technology has been applied in different congenital blood disorders, such as primary immunodeficiencies, X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease or Wiskott-Aldrich syndrome, and inherited bone marrow failure syndromes, such as Fanconi anemia, congenital amegakaryocytic thrombocytopenia or severe congenital neutropenia. Furthermore, CRISPR/Cas9-based gene editing has been implemented successfully as a novel therapy for cancer immunotherapy, by the development of promising strategies such as the use of oncolytic viruses or adoptive cellular therapy to the chimeric antigen receptor-T-cell therapy. Therefore, considering the variety of genes and mutations affected, we can take advantage of the different DNA repair mechanisms by CRISPR/Cas9 in different manners, from homology-directed repair to non-homologous-end-joining to the latest emerging technologies such as base and prime editing. Although the delivery systems into hematopoietic stem and progenitor cells are still the bottleneck of this technology, some of the advances in genome editing shown in this review have already reached a clinical stage and show very promising preliminary results.

CD19 Is Internalized Together with IgM in Proportion to B Cell Receptor Stimulation and Is Modulated by Phosphatidylinositol 3-Kinase in Bone Marrow Immature B Cells.

Newly generated immature B cells that bind self-antigen with high avidity arrest in differentiation and undergo central tolerance via receptor editing and clonal deletion. These autoreactive immature B cells also express low surface levels of the coreceptor CD19, a key activator of the PI3K pathway. Signals emanating from both CD19 and PI3K are known to be critical for attenuating receptor editing and selecting immature B cells into the periphery. However, the mechanisms that modulate CD19 expression at this stage of B cell development have not yet been resolved. Using in vivo and in vitro models, we demonstrate that Cd19 de novo gene transcription and translation do not significantly contribute to the differences in CD19 surface expression in mouse autoreactive and nonautoreactive immature B cells. Instead, CD19 downregulation is induced by BCR stimulation in proportion to BCR engagement, and the remaining surface IgM and CD19 molecules promote intracellular PI3K-AKT activity in proportion to their level of expression. The internalized CD19 is degraded with IgM by the lysosome, but inhibiting lysosome-mediated protein degradation only slightly improves surface CD19. In fact, CD19 is restored only upon Ag removal. Our data also reveal that the PI3K-AKT pathway positively modulates CD19 surface expression in immature B cells via a mechanism that is independent of inhibition of FOXO1 and its role on Cd19 gene transcription while is dependent on mTORC1.

Validity and reliability of the English version of the Diabetic Foot Self-Care Questionnaire: a cross-cultural adaptation.

Introduction: The objective of this study was to carry out the cross-cultural adaptation and validation of the Diabetic Foot Self-Care Questionnaire into the English language, broadening the applicability of this patient-reported outcome measure and improving the monitoring of patients with diabetic foot disease. Methods: The validation study into English was conducted in two phases: cross-cultural adaptation and psychometric validation study. Short Form-12 Version 2, EuroQoL-5D and Foot Function Index were used to analyze the criterion validity. Item response, internal consistency, standard error of measurement, minimal detectable change and construct validity were calculated in the validation phase. Results: An English version of the questionnaire (DFSQ-UMA-En) was successfully obtained. A total of n = 193 participants were tested to confirm the validity and reliability of the questionnaire. Internal consistency values ranged from very good to excellent (Cronbach's α =0.889-0.981), and reliability was excellent (ICC = 0.854-0.959). Standard error measurement value was =2.543. Criterion validity ranged from r = 0.429 to r = 0.844. For construct validity, Kaiser-Meyer-Olkin test was =0.752. Conclusion: DFSQ-UMA-En is a valid and reliable tool with good readability and comprehension features. This questionnaire addresses foot self-care behaviors in patients with diabetic foot disease, standing out as essential for early diagnosis and prevention strategies in clinical and research settings.

Case report: Asymptomatic bronchopulmonary sequestration in an adult with dual celiac and aortic supply.

A rare congenital malformation of the respiratory tract, bronchopulmonary sequestration generally presents in childhood and adolescence with recurrent pneumonia or in adulthood as an incidental finding on thoracic imaging. Manifesting as intrapulmonary or extrapulmonary types, bronchopulmonary sequestration characteristically receives blood supply from the systemic rather than pulmonary circulation. We present a 45-year-old male patient who received a provisional diagnosis of bronchopulmonary sequestration following an incidental finding on routine imaging. This case describes the way in which a provisional diagnosis may be made based upon imaging as well as underscoring the importance of alleviating the burden of additional imaging studies.

Autoantibodies elicited with SARS-CoV-2 infection are linked to alterations in double negative B cells.

Double negative (DN) B cells (CD27-IgD-) comprise a heterogenous population of DN1, DN2, and the recently described DN3 and DN4 subsets. In autoimmune disease, DN2 cells are reported to be precursors to autoreactive antibody secreting cells and expansion of DN2 cells is linked to elevated interferon levels. Severe SARS-CoV-2 infection is characterized by elevated systemic levels of pro-inflammatory cytokines and serum autoantibodies and expansion of the DN2 subset in severe SARS-CoV-2 infection has been reported. However, the activation status, functional capacity and contribution to virally-induced autoantibody production by DN subsets is not established. Here, we validate the finding that severe SARS-CoV-2 infection is associated with a reduction in the frequency of DN1 cells coinciding with an increase in the frequency of DN2 and DN3 cells. We further demonstrate that with severe viral infection DN subsets are at a heightened level of activation, display changes in immunoglobulin class isotype frequency and have functional BCR signaling. Increases in overall systemic inflammation (CRP), as well as specific pro-inflammatory cytokines (TNFα, IL-6, IFNγ, IL-1ß), significantly correlate with the skewing of DN1, DN2 and DN3 subsets during severe SARS-CoV-2 infection. Importantly, the reduction in DN1 cell frequency and expansion of the DN3 population during severe infection significantly correlates with increased levels of serum autoantibodies. Thus, systemic inflammation during SARS-CoV-2 infection drives changes in Double Negative subset frequency, likely impacting their contribution to generation of autoreactive antibodies.

Neutral isocyanide-templated assembly of pillar[5]arene [2] and [3]pseudorotaxanes.

Unprecedented pillar[5]arene-isocyanide pseudorotaxane complexes are reported. Extensive 1H-NMR experiments reveal remarkably strong binding affinities of alkyl diisocyanide guests (Ka > 105 M-1 in CDCl3) by pillar[5]arenes. Characterised by multinuclear 1H and 13C-NMR spectroscopy and single-crystal X-ray diffraction, it is demonstrated that pillar[5]arenes are capable of encapsulating a series of alkyl diisocyanides wherein either [2]- or [3]pseudorotaxanes can be formed by varying the alkyl chain length. Moreover, electron-deficient aryl isocyanides, are demonstrated to form inclusion complexes within the cavities of pillar[5]arenes stabilised by multiple C-H⋯π interactions.

BRAF Modulates Lipid Use and Accumulation.

There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.