Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8–17% of patients with epilepsy. Although the mechanisms of SUDEP are essentially unknown, one proposed mechanism is respiratory arrest initiated by a convulsive seizure. In mice, we have previously observed that extended apnea occurs during the tonic phase of seizures. Although often survived, tonic seizures became fatal when breathing did not immediately recover postictally. We also found that respiratory muscles were tonically contracted during the apnea, suggesting that muscle contraction could be the cause of apnea. In the present study, we tested the hypothesis that pyramidal neurons of the motor cortex drive motor units during the tonic phase, which produces apnea. Mice harboring the patient-derived N1768D point mutation of an Scn8a allele were crossed with transgenic mice such that inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADD) receptors were selectively expressed in excitatory forebrain neurons. We then triggered audiogenic and hippocampal (HC) stimulated seizures under control conditions and when excitatory forebrain neurons were inhibited with the synthetic ligand Clozapine-N-Oxide (CNO). We found that inhibition with CNO was sufficient to increase seizure threshold of HC stimulated, but not audiogenic, seizures. In addition, regardless of seizure type, CNO nearly eliminated epileptiform activity that occurred proximal to the tonic phase; however, the seizure behaviors, notably the tonic phase and concomitant apnea, were unchanged. We interpret these results to indicate that while cortical neurons are likely critical for epileptogenesis and seizure initiation, the behavioral manifestations of tonic seizures are generated by neural circuitry in the mid- and/or hindbrain.

BackgroundStudies of human patients have shown that most anti-RBC alloantibodies are IgG1 or IgG3 subclasses, though it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation.Study Design and MethodsWT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA.ResultsWhen compared to antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination.DiscussionOur results show that anti-RBC class-switching occurs via alternate mechanisms when compared to the well-studied immunogen alum vaccination.

Background Studies of human patients have shown that most anti‐RBC alloantibodies are IgG1 or IgG3 subclasses, although it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class‐switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation. Study Design and Methods WT mice were either immunized with Alum/HEL‐OVA or transfused with HOD RBCs and levels of anti‐HEL IgG subtypes were measured using end‐point dilution ELISAs. To study the role of STAT6 in IgG class‐switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL‐OVA, and IgG subclasses were quantified by ELISA. Results When compared with antibody responses to Alum/HEL‐OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b, and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination. Discussion Our results show that anti‐RBC class‐switching occurs via alternate mechanisms when compared with the well‐studied immunogen alum vaccination.

Sudden Unexpected Death in Epilepsy (SUDEP) is defined as the sudden, unexpected and unexplained death of a person with epilepsy and accounts for between 8 and 17% of epilepsy‐related deaths, rising to 50% for patients with refractory epilepsy. In a mouse model of SUDEP, we have recently shown that death is due to seizure‐induced respiratory arrest. In addition, apnea is initiated during the tonic phase and tonic respiratory muscle contraction is a possible mechanism of apnea. In the present study, we explore 1) whether tonic activity of the inspiratory rhythm generator in the brainstem and/or 2) upper motor neuron activity in the motor cortex drives ictal apnea. We recorded video, electrocorticogram (ECoG), electrocardiogram (ECG), and breathing via whole body plethysmography in adult mice carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; “D/+ mice”) during audiogenic seizures. This mutation was identified from a patient that died from SUDEP and D/+ mice have severe convulsive seizures with apnea and many suffer seizure‐induced death. To test the hypothesis that tonic activity from the inspiratory oscillator results in apnea, we implanted fiberoptic ferrules bilaterally into the Bötzinger Complex (BötC) of mice that express Channelrhodopsin2 (ChR2) under the vesicular GABA transporter (VGAT; “VGAT‐ChR2” mice) that were crossed with D/+ mice (Fig. 1A & B). The goal of the experiment is to photostimulate BötC during ictal apnea to inhibit tonic inspiratory activity and produce expiration (Fig. 1C). Seizures were evoked using a 15 kHz pure tone, as we have done before (Fig. 1D). Trains of light pulses (50 ms pulses, 5 mW of 473 nm light) were evoked repetitively during ictal apnea (Fig. 1E & F). However, this did not recover normal breathing rhythm and apnea duration was no different for any photostimulation paradigm versus control (p = 0.7892, F = 0.1747, One‐Way ANOVA; Fig. 1G). Although breathing was not affected during seizures, the effects on baseline breathing were substantial; for example, inspiration was inhibited for a full 10 second photostimulation train (Fig. 1H). To test the necessity of ictal activity from upper motor neurons in the motor cortex are required for generating ictal apnea, we expressed iDREADD receptors in cortical excitatory neurons of D/+ mice and injected CNO i.p. prior to inducing seizures with a 15 kHz pure tone (Fig. 2A). Under control conditions, seizures presented with the usual tonic phase apnea and spike wave discharges (SWDs) in the motor cortex (Fig. 2B). CNO was able to robustly inhibit the SWDs, but the tonic phase and apnea were not affected (Figs. 2C‐E). The effect of 3 mg/kg CNO on ECoG power was significantly greater than the effect on apnea (p = 0.0059, paired t‐test). In sum, we found that the core inspiratory oscillator circuitry in the brainstem is likely bypassed to create tonic inspiratory activity. Furthermore, inhibition of cortical upper motor neurons has no effect on apnea. Thus, our interpretation is that other pools of upper motor neurons...