Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis.

Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

CARD19, a Novel Regulator of the TAK1/NF-κB Pathway in Self-Reactive B Cells.

The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-ß-activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance.

A lineage-specific requirement for YY1 Polycomb Group protein function in early T cell development.

Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group protein (PcG) with important functions for regulating lymphocyte development and stem cell self-renewal. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that result in histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in the hematopoietic system results in an early T cell developmental blockage at the double negative (DN) 1 stage with reduced Notch1 signaling. There is a lineage-specific requirement for YY1 PcG function. YY1 PcG domain is required for T and B cell development but not necessary for myeloid cells. YY1 functions in early T cell development are multicomponent and involve both PcG-dependent and -independent regulations. Although YY1 promotes early T cell survival through its PcG function, its function to promote the DN1-to-DN2 transition and Notch1 expression and signaling is independent of its PcG function. Our results reveal how a ubiquitously expressed PcG protein mediates lineage-specific and context-specific functions to control early T cell development.

Exosomes from human urine-derived stem cells carry NRF1 to alleviate bladder fibrosis via regulating miR-301b-3p/TGFßR1 pathway.

Bladder outlet obstruction (BOO) is a common disease that always make the bladder develops from inflammation to fibrosis. This study was to investigate the effect of exosomes from human urine-derived stem cells (hUSCs) on bladder fibrosis after BOO and the underlying mechanism. The BOO mouse model was established by inserting a transurethral catheter, ligation of periurethral wire, and removal of the catheter. Mouse primary bladder smooth muscle cells (BSMCs) were isolated and treated with TGFß1 to mimic the bladder fibrosis model in vitro. Exosomes from hUSCs (hUSC-Exos) were injected into the bladder of BOO mice and added into the culture of TGFß1-induced BSMCs. The associated factors in mouse bladder tissues and BSMCs were detected. It was confirmed that the treatment of hUSC-Exos alleviated mouse bladder fibrosis and down-regulated fibrotic markers (a-SMA and collagen III) in bladder tissues and TGFß1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFßR1 was a target of NRF1 and miR-301b-3p, and miR-301b-3p was a target of NRF1. It was next characterized that hUSC-Exos carried NRF1 to up-regulate miR-301B-3p, thereby reducing TGFßR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFßR1 pathway.

Phenylacetyl glutamine: a novel biomarker for stroke recurrence warning.

BACKGROUND: Stroke is the second leading cause of disease-related death and the third leading cause of disability worldwide. However, how to accurately warn of stroke onset remains extremely challenging. Recently, phenylacetyl glutamine (PAGln) has been implicated in the onset of stroke, but evidences from cohort studies of onset are lacking, especially in patients with first-onset or recurrent. It is necessary to deeply demonstrate the effectiveness of PAGln level on warning stroke onset. METHODS: One hundred fifteen first onset stroke patients, 33 recurrent stroke patients, and 135 non-stroke controls were included in the analysis. Risk factors associated with stroke attacking were evaluated, and plasma PAGln levels were detected via HPLC-MS based method. LASSO regression, Pearson correlation analysis, and univariate analysis were carried out to demonstrate the associations between PAGln levels and risk factors of stroke. Random forest machine learning algorithm was used to build classification models to achieve the distinction of first-onset stroke patients, recurrent stroke patients, and non-stroke controls, and further demonstrate the contribution of PAGln levels in the distinction of stroke onset. RESULTS: The median level of PAGln in the first-onset stroke group, recurrent stroke group, and non-stroke group was 933 ng/mL, 1014 ng/mL, and 556 ng/mL, respectively. No statistical correlation was found between PAGln level and subject's living habits, eating preferences, and concomitant diseases (hypertension, hyperlipidemia, and diabetes). Stroke severity indicators, mainly age and NIHSS score, were found associate with the PAGln levels. Machine learning classification models confirmed that PAGln levels, as the main contributing variable, could be used to distinguish recurrent stroke patients (but not first-onset stroke patients) from non-stroke controls. CONCLUSION: PAGln may be an effective indicator to monitor the recurrence in stroke patients.

Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity.

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

Generation of PECAM-1 (CD31) conditional knockout mice.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM-1 functions, we generated mice in which PECAM1, the gene encoding PECAM-1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3' loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT-flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 flox/flox offspring, which expressed PECAM-1 at normal levels and had no overt phenotype. PECAM1 flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY-box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 del/WT ), which were crossbred to generate Sox2Cre; PECAM1 del/del offspring. Sox2Cre; PECAM1 del/del mice recapitulated the phenotype of conventional global PECAM-1 knockout mice. PECAM1 flox/flox mice will be useful for studying distinct roles of PECAM-1 in tissue specific contexts and to gain insights into the roles that PECAM-1 plays in blood and vascular cell function.

Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development.

Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca2+ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca2+ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

Kras Is Critical for B Cell Lymphopoiesis.

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell-specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre-B cell stage and late B cell maturation, resulting in the reduction of BM pre-, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell-specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre-BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre-B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Phospholipase Cγ2 plays a role in TCR signal transduction and T cell selection.

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγ1 and PLCγ2. It is known that PLCγ1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγ1/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγ1 single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγ1/PLCγ2 double-deficient T cells compared with PLCγ1 single-deficient T cells. TCR-mediated signal transduction, including Ca²âº mobilization and Erk activation, was further impaired in PLCγ1/PLCγ2 double-deficient relative to PLCγ1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγ1 heterozygous- and PLCγ2 homozygous-deficient (PLCγ1⁺/⁻PLCγ2⁻/⁻) relative to wild-type, PLCγ2 single-deficient (PLCγ2⁻/⁻), or PLCγ1 heterozygous-deficient (PLCγ1⁺/⁻) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection.

Critical role of B cell lymphoma 10 in BAFF-regulated NF-κB activation and survival of anergic B cells.

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase ß, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

Cerebral venous thrombosis presented with symmetrical crescent-shaped intracranial hemorrhage in alcoholic liver disease: Case reports.

RATIONALE: Cerebral venous thrombosis (CVT) is a relatively uncommon but fatal disease. It can be caused by a variety of hereditary or acquired thrombotic diseases. Initial presentation with intracranial hemorrhage (ICH) in CVT is rare but can further complicate the therapeutic measures and prognosis. Cases of CVT presented with ICH in patients with alcoholic liver disease (ALD) have not been described in the literature, and it might be related with hemostatic abnormalities in ALD patients. PATIENT CONCERNS: We report 2 cases of men admitted to our hospital who were diagnosed with CVT but initially presented with symmetrical crescent-shaped ICH; both of them were ALD patients. DIAGNOSES: Cerebral imaging revealed extended CVT in both cases. The first case was a 64-year-old man with ALD deteriorated with unconsciousness and convulsions; computed tomography showed symmetrical crescent-shaped ICH in the right temporal lobe, and magnetic resonance venography revealed CVT. Another 50-year-old man with ALD complained about dizziness and weakness of his right limbs; computed tomography revealed symmetrical crescent-shaped ICH in bilateral parietal and occipital lobes, and magnetic resonance venography revealed CVT. INTERVENTIONS: The first patient was referred to the endovascular thrombectomy. Both of them were treated with anticoagulation treatment. OUTCOMES: Favorable outcomes were observed in both patients. LESSONS: Symmetrical or multiple crescent-shaped ICH requires a high suspicion in the diagnosis of CVT; even with hemorrhage, it is still important to initiate anticoagulation therapy promptly. The crescent-shaped ICH might be a new sign for CVT, and further studies are needed in the underlying mechanisms of ALD and potential thrombophilia.

Case report: Successful treatment of an anti-D2R and DPPX antibody-associated autoimmune encephalitis patient with high-dose methylprednisolone and intravenous immunoglobulin.

Background: Autoimmune encephalitis is a neurological condition caused by abnormal immune responses, manifesting as cognitive impairments, behavioral abnormalities, and seizures. Its diagnosis depends on the detecting neuronal surface antibodies in serum or cerebrospinal fluid. Despite recent advances in understanding, clinical recognition remains challenging, especially with rare antibodies such as anti-dopamine D2 receptor (D2R) and anti-dipeptidyl-peptidase-like protein 6 (DPPX) antibodies. Delayed diagnosis can lead to severe complications. This case presentation emphasizes the diagnostic intricacies and effective treatment of the anti-D2R and DPPX antibody-associated autoimmune encephalitis. Case description: The patient presented with a 3-day history of fatigue and limb soreness followed by a 3-h episode of confusion and limb convulsions. Upon admission to our facility, the initial diagnosis included status epilepticus, aspiration pneumonia, metabolic acidosis, respiratory alkalosis, and suspected encephalitis. Despite receiving antiepileptic, anti-infection, and antivirus therapy, the patient's condition deteriorated. Both computed tomography (CT) scan and magnetic resonance imaging (MRI) of the brain showed no significant abnormalities. No pathogen was identified in the cerebrospinal fluid (CSF). However, further CSF and serum examination revealed positive results of anti-D2R and anti-DPPX antibodies, confirming a diagnosis of anti-D2R and DPPX antibody-associated autoimmune encephalitis. The patient underwent a comprehensive treatment regimen, including high-dose methylprednisolone pulse therapy combined with intravenous immunoglobulin (IVIG), antiviral and anti-infection treatments, and antiepileptic medications. Significant clinical improvement was observed, and by the 18th day of admission, the patient was stable and coherent. Conclusions: The current patient represents the first reported case of double-positive autoimmune encephalitis for anti-D2R and DPPX antibodies, with epilepsy as a prominent feature. High-dose methylprednisolone pulse therapy combined with IVIG has shown significant safety and efficacy in treating anti-D2R and DPPX antibody-positive autoimmune encephalitis-associated epilepsy.

Phosphorylation of Bcl10 negatively regulates T-cell receptor-mediated NF-kappaB activation.

Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation.

Essential role of phospholipase C gamma 2 in early B-cell development and Myc-mediated lymphomagenesis.

Phospholipase Cgamma2 (PLCgamma2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCgamma2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCgamma2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCgamma2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCgamma2(-/-) Emu-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Emu-Myc transgenics. Furthermore, lymphomas from PLCgamma2(-/-) Emu-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCgamma2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

CXCR5+PD-1+ follicular helper CD8 T cells control B cell tolerance.

Many autoimmune diseases are characterized by the production of autoantibodies. The current view is that CD4+ T follicular helper (Tfh) cells are the main subset regulating autoreactive B cells. Here we report a CXCR5+PD1+ Tfh subset of CD8+ T cells whose development and function are negatively modulated by Stat5. These CD8+ Tfh cells regulate the germinal center B cell response and control autoantibody production, as deficiency of Stat5 in CD8 T cells leads to an increase of CD8+ Tfh cells, resulting in the breakdown of B cell tolerance and concomitant autoantibody production. CD8+ Tfh cells share similar gene signatures with CD4+ Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance.

Gab2 and Gab3 Redundantly Suppress Colitis by Modulating Macrophage and CD8+ T-Cell Activation.

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8+ T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3-/-) were generated. Gab2/3-/- mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3-/- hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo. In spontaneous disease, intestinal intraepithelial CD8+ but much fewer CD4+, T-cells from Gab2/3-/- mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8+ T-cell activation and suppress chronic colitis.