Citations

Abstract The circadian clock imposes a critical yet incompletely understood layer of regulation on adaptive immunity. T helper 17 (Th17) antimicrobial immune responses including expression of IL-17A by lamina propria CD4⁺ T cells, exhibit diurnal variation and are sensitive to circadian disruption. While the core circadian regulators CLOCK and BMAL1 canonically function as a heterodimer to drive rhythmic gene expression, emerging evidence suggests they may have distinct regulatory roles. Here we integrated ChIP-seq, ATAC-seq, and RNA-seq analyses in naïve CD4⁺ T cells to reveal that the CLOCK-BMAL1 complex controls circadian and metabolic programs through promoter binding, whereas exclusive CLOCK binding at promoters, together with CLOCK-BMAL1 binding at enhancers, regulates immune-associated genes. Using the mutant CLOCKΔ19, which lacks the transactivation domain, we observed disrupted circadian transcription and globally reduced chromatin accessibility, alongside increased accessibility at Th17-associated loci that their altered temporal regulation. Functionally, ClockΔ19 produces β-catenin stabilization in T cells, pronounced expansion of RORγt⁺ CD4⁺ T cells and Treg cells, impaired Treg suppression of Th17 responses, heightened Th17 responses, and reduced IFN-γ production during viral infection. Collectively, these findings define BMAL1 dependent and independent CLOCK functions that program naïve CD4⁺ T cell fate, restrain Th17 differentiation, and preserve immune homeostasis.

AbstractBackgroundClot based assays used for lupus anticoagulant (LAC) detection are typically interpreted in a qualitative fashion and may not reflect LAC potency. In this cross‐sectional study, we describe a method for quantifying the LAC titer using serial (dependent) two‐fold dilutions in normal pooled plasma.MethodsSerial dilutions of 51 residual plasma samples from 50 patients were tested using the Russell's viper venom screening time (DRVVT) and activated partial thromboplastin screening time (APTT) methodologies. The measured clotting times and the corresponding dilution factors were then used to derive a four‐parameter logistic model. The LAC titer for each patient was interpolated as the sample dilution that corresponds to the upper reference interval limit of the corresponding assay.ResultsCalculated APTT and DRVVT LAC titers displayed a strong linear correlation (R2 = 0.84) between each other, but not with the degree of prolongation of the APTT/DRVVT screening time in the neat undiluted samples. Using data driven partitioning, patients could be grouped into low (<10) or high (≥10) DRVVT LAC titer. There were no significant differences in anticardiolipin (aCL) or anti‐beta 2 glycoprotein 1 (aB2GPI) antibody levels or prevalence of thromboembolic events between low and high LAC titer groups. In contrast, antiphosphatidylserine/prothrombin (aPS/PT) IgM antibody levels, but not IgG, were significantly higher in the high LAC titer group.ConclusionsThe degree of prolongation of the APTT/DRVVT screening time is not correlated with the LAC titer. Only aPS/PT IgM antibodies levels were strongly correlated with the LAC titers. Additional studies are warranted to determine clinical implications of high LAC titers.

Abstract Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but whether the circadian clock regulates the adaptive immune response to cancer is mostly unclear. In mouse models of polyposis, and human colorectal cancer patients, we showed that tumor infiltrating Tregs overexpressed b-catenin and RORgt, and secreted IL-17. These Tregs suppressed tumor reactive cytotoxic T cells, and promoted tumor growth. Therefore, we examined how the expression of a dominant negative ClockΔ19 gene affects inflammation and susceptibility to cancer in the APCΔ468 mouse model of hereditary polyposis. ClockΔ19 mutant mice had high expression of RORgt+ and b-catenin+ Tregs. These mice showed a notable expansion of Th17 inflammation in vivo upon injection of aCD3. Functionally these Tregs showed pro-inflammatory properties. In addition to these effects, APCΔ468 ClockΔ19 mice had increased numbers of CD11b+ cell, Ly6C+CXCR1− monocytes, and Ly6C+Ly6G+ myeloid derived suppressor cells (MDSC) as compared with APCΔ468 mice. APCΔ468 ClockΔ19 mice had higher tumor burden in the small intestine and colon than the APCΔ468 mice. Both T-cells and tumors had intense stabilization of nuclear b-catenin and were more aggressive as indicated by abundance of stromal mMCP6+ mast cells (MC), which typically appear at the invasive borders of tumors. Our finding indicating increase in tumor load and worsening inflammation caused by direct ClockΔ19 effects in the epithelium and enhanced TH17 bias in the immune system.

Abstract Regulatory T-cells (Tregs) play a dual role in colon cancer, promoting immune tolerance and suppressing inflammation. Two types of Tregs infiltrate the gut. Thymic derived Helios+CD4+Foxp3+ Tregs have high affinity for self-antigens, including tumor-antigens and protect the tumor, while Helios−CD4+Foxp3+ extra-thymic Tregs that are generated in the gut have microbial specificity and suppress harmful inflammation. In colon cancer, there is a preferential expansion of a population of Helios−CD4+Foxp3+RORγt+ Tregs with enhanced T-cell suppressive properties and pro-inflammatory properties that promote tumor growth. We showed that this population of Tregs in cancer patients expresses elevated levels of β-catenin. To test the role of canonical Wnt signaling in the generation of pro-inflammatory Tregs, we stabilized β-catenin or deleted its DNA binding partner TCF-1 in Tregs. In both instances expression of RORγt was upregulated. While the stabilization of β-catenin led to loss of Treg functions, ablation of TCF-1 reproduced the phenotype and functional characteristics of Tregs in colon cancer. The TCF-1 deficient Tregs were pro-inflammatory, potent T-cell suppressive, and tumor promoting.

AbstractVon Willebrand factor (VWF) is a multimeric glycoprotein with essential roles in primary hemostasis. Patients with von Willebrand disease (VWD), due to quantitative and/or qualitative defects of VWF usually experience mucocutaneous bleeding. Based on the laboratory results of VWF antigen, various VWF activities, factor VIII activity, and VWF multimer patterns, VWD can be categorized as type 1, 2, and 3 VWD. VWF multimer analysis by either manual or semi-automated electrophoresis and immunoblotting is a critical part of the laboratory testing to differentiate type 1, type 2 VWD, and subtypes of type 1 or 2 VWD. The multimer distribution patterns can also help to understand the underlying molecular mechanism of VWF synthesis, multimerization, and clearance defects in VWD. This review will cover VWF synthesis, multimerization, secretion, VWF multimer analysis, and VWF multimer interpretation of various types and subtypes of VWD.

Abstract Background: Heparin induced thrombocytopenia (HIT) is a rare immune mediated complication that is triggered in a subset of patients temporal to therapeutic heparin exposure. Laboratory testing is based on screening for the presence of serum anti-PF4 antibodies using sensitive solid-phase immunoassays. If antibodies are detected, functional testing to demonstrate the platelet activating properties and heparin dependence of these immune complexes is then performed. Previous studies have reported possible clinical utility in identifying non heparin dependent platelet activating antibodies using a buffer control with zero heparin in the serotonin release functional assay (SRA). These reports suggested a correlation between reactivity in the zero heparin buffer control and pathogenicity of HIT antibodies which may define a subtype of HIT, referred to as autoimmune HIT. We aimed to investigate the utility of zero heparin buffer control as a part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA). Methods: Three hundred archived serum samples were tested using anti-PF4 IgG antibody enzyme-linked immunosorbent assay (ELISA; Immucor Diagnostics, GA, USA). SRA was preformed on all samples using Mayo-SRA and reference 14C SRA methods. Zero heparin control buffer was included in the Mayo-SRA assay. Serotonin release &gt;20% in the low dose heparin (0.1U/mL, LDH) and ELISA optical density (OD) &gt;0.4 were considered positive. Drug interference studies were performed by spiking known SRA-positive samples with increasing concentrations of unfractionated heparin (UFH), low molecular weight heparin (LMWH) or fondaparinux in the LDH and zero heparin SRA. The clinical 4T score was calculated retrospectively calculated for all patients. Results: Of the 300 tested samples, 57 were anti-PF4 ELISA positive. 33 of the 57 samples were positive using the reference 14C SRA method. Whereas 43 samples were positive by Mayo-SRA assay. Three additional samples were positive by Mayo-SRA, but negative by both screening anti-PF4 ELISA and the reference SRA method (Fig- 1A). Lastly, 13 samples were anti-PF4 ELISA positive, but SRA negative by both methods in comparison. Interestingly, 44 of 46 (95%) samples interpreted positive by LDH were also interpreted positive (serotonin release &gt;20%) under zero heparin conditions. These included the 3 samples that were positive by Mayo-SRA but negative by both screening anti-PF4 ELISA and the reference SRA. The overall % serotonin release using zero heparin control was significantly lower (P= 0.003, paired Student T-test) compared to LDH (Fig-1B). In addition, zero heparin followed a similar pattern as LDH, with highest levels at ELISA OD units &gt;2 (Fig-1C). Strikingly, drug interference studies showed artifactual serotonin release in the zero heparin reaction, which was not detected in the absence of spiked drugs. For UFH, serotonin release in zero heparin control occurred at very low spiked concentrations, ≥0.063 U/mL. LMWH and fondaparinux spiking experiments also displayed similar zero heparin serotonin release patterns (Fig-1D). Of note, none of these interferences were detected in UFH spiked SRA-negative samples (data not shown). Conclusion: Contrary to prior results suggesting that less than 50% of LDH SRA positive samples are also positive in the zero heparin SRA, our results show high zero heparin SRA positivity rate of &gt;95%. Zero heparin SRA showed a pattern with highest levels at ELISA OD units &gt;2 suggesting that reactivity in this condition is a function of antibody strength rather than a qualitative difference (i.e. "autoimmune" HIT antibodies vs "non-autoimmune" HIT antibodies). In addition, contamination of patient sera with small amounts of remnant heparin can significantly impact platelet activation in the zero heparin SRA test. Thus, zero heparin SRA positive results may be artifactual and represent residual heparin contained in the patient sample. Figure legend: Fig-1A. Scatter plots of SRA positive samples grouped by 4T scores. Red dots are Mayo-SRA only positive samples. Fig-1B and Fig-1C. Scatter plots of % serotonin release of Mayo-SRA positive samples at LDH (circles) vs. zero heparin buffers (squares), and grouped by ELISA OD values, respectively. Fig-1D. % Serotonin release of known SRA-positive samples spiked with increasing concentrations of UFH, LMWH, or fondaparinux at LDH (white) or zero heparin buffers (red). Figure 1 Figure 1. Disclosures Pruthi: CSL Behring: Honoraria; Genentech: Honoraria; Bayer Healthcare AG: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.