Opposing T cell responses in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Hippocampal sclerosis in temporal lobe epilepsy: findings at 7 T¹.

PURPOSE: To determine if ultrahigh-field-strength magnetic resonance (MR) imaging can be used to detect subregional hippocampal alterations. MATERIALS AND METHODS: Subjects provided written consent to participate in this prospective institutional review board-approved HIPAA-compliant study. T1- and T2-weighted 7-T brain MR images were acquired in 11 healthy subjects and eight patients with temporal lobe epilepsy (TLE). In all subjects, images were qualitatively examined for evidence of hippocampal atrophy, signal change, and malrotation with the Bernasconi definition, and digitations of the hippocampal heads were counted (agreement was measured with the κ statistic). Data were analyzed quantitatively with manual subregional hippocampal body segmentation. Subregional data in individual subjects with TLE were compared with data in control subjects to detect deviation from the control range for volume measures on each side and with asymmetry indexes. RESULTS: All eight patients with TLE had hippocampal abnormalities on the epileptogenic side. Subregional analysis revealed selective lateral Ammon horn atrophy in six patients and diffuse Ammon horn and dentate gyrus atrophy in one patient. Paucity of hippocampal digitations occurred on the epileptogenic side in all patients with TLE and also on the contralateral side in three patients (interrater κ value, 0.80). Hippocampal malrotation was observed in three patients with TLE and four control subjects. CONCLUSION: Ultrahigh-field-strength MR imaging permitted detection of selectively greater Ammon horn atrophy in patients with TLE and hippocampal sclerosis. Paucity of digitations is a deformity of the hippocampal head that was detected independent of hippocampal atrophy in patients with mesial TLE.

Human KIR + CD8 + T cells target pathogenic T cells in Celiac disease and are active in autoimmune diseases and COVID-19.

Previous reports show that Ly49 + CD8 + T cells can suppress autoimmunity in mouse models of autoimmune diseases. Here we find a markedly increased frequency of CD8 + T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various autoimmune diseases. Moreover, KIR + CD8 + T cells can efficiently eliminate pathogenic gliadin-specific CD4 + T cells from Celiac disease (CeD) patients' leukocytes in vitro . Furthermore, we observe elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Expanded KIR + CD8 + T cells from these different diseases display shared phenotypes and similar T cell receptor sequences. These results characterize a regulatory CD8 + T cell subset in humans, broadly active in both autoimmune and infectious diseases, which we hypothesize functions to control self-reactive or otherwise pathogenic T cells. ONE-SENTENCE SUMMARY: Here we identified KIR + CD8 + T cells as a regulatory CD8 + T cell subset in humans that suppresses self-reactive or otherwise pathogenic CD4 + T cells.

A Circulating Bioreactor Reprograms Cancer Cells Toward a More Mesenchymal Niche.

Cancer is a complex and heterogeneous disease, and cancer cells dynamically interact with the mechanical microenvironment such as hydrostatic pressure, fluid shear, and interstitial flow. These factors play an essential role in cell fate and circulating tumor cell heterogeneity, and can influence the cellular phenotype. In this study, a peristaltic continuous flow reactor is designed and applied to HCT-116 colorectal carcinoma cells to mimic the fluid dynamics of circulation. With this intervention, a CD44/CD24-cell subpopulation emerges, and 100 genes are significantly regulated. The expression of cells at 4 h in the flow reactor is very similar to TGF-ß treatment, which is an inducer of epithelial-mesenchymal transition. ATF3 and SERPINE1 are significantly upregulated in these groups, suggesting that the mesenchymal transition is induced through this signaling pathway. This flow reactor model is satisfactory on its own to reprogram colorectal cancer cells toward a more mesenchymal niche mimicking circulation of the blood.

Quantitative analysis and biophysically realistic neural modeling of the MEG mu rhythm: rhythmogenesis and modulation of sensory-evoked responses.

Variations in cortical oscillations in the alpha (7-14 Hz) and beta (15-29 Hz) range have been correlated with attention, working memory, and stimulus detection. The mu rhythm recorded with magnetoencephalography (MEG) is a prominent oscillation generated by Rolandic cortex containing alpha and beta bands. Despite its prominence, the neural mechanisms regulating mu are unknown. We characterized the ongoing MEG mu rhythm from a localized source in the finger representation of primary somatosensory (SI) cortex. Subjects showed variation in the relative expression of mu-alpha or mu-beta, which were nonoverlapping for roughly 50% of their respective durations on single trials. To delineate the origins of this rhythm, a biophysically principled computational neural model of SI was developed, with distinct laminae, inhibitory and excitatory neurons, and feedforward (FF, representative of lemniscal thalamic drive) and feedback (FB, representative of higher-order cortical drive or input from nonlemniscal thalamic nuclei) inputs defined by the laminar location of their postsynaptic effects. The mu-alpha component was accurately modeled by rhythmic FF input at approximately 10-Hz. The mu-beta component was accurately modeled by the addition of approximately 10-Hz FB input that was nearly synchronous with the FF input. The relative dominance of these two frequencies depended on the delay between FF and FB drives, their relative input strengths, and stochastic changes in these variables. The model also reproduced key features of the impact of high prestimulus mu power on peaks in SI-evoked activity. For stimuli presented during high mu power, the model predicted enhancement in an initial evoked peak and decreased subsequent deflections. In agreement, the MEG-evoked responses showed an enhanced initial peak and a trend to smaller subsequent peaks. These data provide new information on the dynamics of the mu rhythm in humans and the model provides a novel mechanistic interpretation of this rhythm and its functional significance.

A computational model of the ribbon synapse.

A model of the ribbon synapse was developed to replicate both pre- and postsynaptic functions of this glutamatergic juncture. The presynaptic portion of the model is rich in anatomical and physiological detail and includes multiple release sites for each ribbon based on anatomical studies of presynaptic terminals, presynaptic voltage at the terminal, the activation of voltage-gated calcium channels and a calcium-dependent release mechanism whose rate varies as a function of the calcium concentration that is monitored at two different sites which control both an ultrafast, docked pool of vesicles and a release ready pool of tethered vesicles. The postsynaptic portion of the program models diffusion of glutamate and the physiological properties of glutamatergic neurotransmission in target cells. We demonstrate the behavior of the model using the retinal bipolar cell to ganglion cell ribbon synapse. The model was constrained by the anatomy of salamander bipolar terminals based on the ultrastructure of these synapses and presynaptic contacts were placed onto realistic ganglion cell morphology activated by a range of ribbon synapses (46-138). These inputs could excite the cell in a manner consistent with physiological observations. This model is a comprehensive, first-generation attempt to assemble our present understanding of the ribbon synapse into a domain that permits testing our understanding of this important structure. We believe that with minor modifications of this model, it can be fine tuned for other ribbon synapses.

Modeling the contrasting Neolithic male lineage expansions in Europe and Africa.

BACKGROUND: Patterns of genetic variation in a population carry information about the prehistory of the population, and for the human Y chromosome an especially informative phylogenetic tree has previously been constructed from fully-sequenced chromosomes. This revealed contrasting bifurcating and starlike phylogenies for the major lineages associated with the Neolithic expansions in sub-Saharan Africa and Western Europe, respectively. RESULTS: We used coalescent simulations to investigate the range of demographic models most likely to produce the phylogenetic structures observed in Africa and Europe, assessing the starting and ending genetic effective population sizes, duration of the expansion, and time when expansion ended. The best-fitting models in Africa and Europe are very different. In Africa, the expansion took about 12 thousand years, ending very recently; it started from approximately 40 men and numbers expanded approximately 50-fold. In Europe, the expansion was much more rapid, taking only a few generations and occurring as soon as the major R1b lineage entered Europe; it started from just one to three men, whose numbers expanded more than a thousandfold. CONCLUSIONS: Although highly simplified, the demographic model we have used captures key elements of the differences between the male Neolithic expansions in Africa and Europe, and is consistent with archaeological findings.

An assessment of an educational intervention on resident physician attitudes, knowledge, and skills related to adverse event reporting.

OBJECTIVE: Reporting and learning from events linked to patient harm and unsafe conditions is critical to improving patient safety. Programs that engage resident physicians in adverse event reporting can enhance patient safety and simultaneously address all 6 Accreditation Council for Graduate Medical Education competencies. Yet fewer than 60% of physicians know how to report adverse events and near misses, and fewer than 40% know what to report. Our study evaluated the effect of an educational intervention on anesthesiology residents' attitudes, knowledge, and skills related to adverse event reporting and the associated follow-up. METHODS: In a prospective study, anesthesiology residents participated in a training program focused on the importance of reporting methods and on reporting adverse events for patient safety. Quarterly adverse event reports were analyzed retrospectively for 2 years before the intervention and prospectively for 7 quarters after the intervention. Residents also completed a survey, before and 1 year after the intervention, that evaluated their attitudes, experience, and knowledge regarding adverse event reporting. RESULTS: After the intervention, the number of adverse event reports increased from 0 per quarter to almost 30 per quarter. We identified several categories of harm events, near misses, and unsafe conditions, including reports of disruptive providers. Of the harm events associated with invasive procedures, more than half were associated with lack of attending physician supervision. We also observed significant progress in the residents' ability to appropriately file a report, improved attitudes regarding the value of reporting and available emotional support, and a reduction in the perceived impediments to reporting. CONCLUSIONS: An educational intervention increased the number of adverse event reports submitted by anesthesiology residents, improved their attitudes about the importance of reporting, and produced a source for learning opportunities and process improvements in the delivery of anesthesia care.

Retinal bipolar cells: temporal filtering of signals from cone photoreceptors.

The temporal dynamics of the response of neurons in the outer retina were investigated by intracellular recording from cones, bipolar, and horizontal cells in the intact, light-adapted retina of the tiger salamander (Ambystoma tigrinum), with special emphasis on comparing the two major classes of bipolars cells, the ON depolarizing bipolars (Bd) and the OFF hyperpolarizing bipolars (Bh). Transfer functions were computed from impulse responses evoked by a brief light flash on a steady background of 20 cd/m(2). Phase delays ranged from about 89 ms for cones to 170 ms for Bd cells, yielding delays relative to that of cones of about 49 ms for Bh cells and 81 ms for Bd cells. The difference between Bd and Bh cells, which may be due to a delay introduced by the second messenger G-protein pathway unique to Bd cells, was further quantified by latency measurements and responses to white noise. The amplitude transfer functions of the outer retinal neurons varied with light adaptation in qualitative agreement with results for other vertebrates and human vision. The transfer functions at 20 cd/m(2) were predominantly low pass with 10-fold attenuation at about 13, 14, 9.1, and 7.7 Hz for cones, horizontal, Bh, and Bd cells, respectively. The transfer function from the cone voltage to the bipolar voltage response, as computed from the above measurements, was low pass and approximated by a cascade of three low pass RC filters ("leaky integrators"). These results for cone-->bipolar transmission are surprisingly similar to recent results for rod-->bipolar transmission in salamander slice preparations. These and other findings suggest that the rate of vesicle replenishment rather than the rate of release may be a common factor shaping synaptic signal transmission from rods and cones to bipolar cells.

Natural images and contrast encoding in bipolar cells in the retina of the land- and aquatic-phase tiger salamander.

Intracellular recordings were obtained from 57 cone-driven bipolar cells in the light-adapted retina of the land-phase (adult) tiger salamander (Ambystoma tigrinum). Responses to flashes of negative and positive contrast for centered spots of optimum spatial dimensions were analyzed as a function of contrast magnitude. On average, the contrast/response curves of depolarizing and hyperpolarizing bipolar cells in the land-phase animals were remarkably similar to those of aquatic-phase animals. Thus, the primary retinal mechanisms mediating contrast coding in the outer retina are conserved as the salamander evolves from the aquatic to the land phase. To evaluate contrast encoding in the context of natural environments, the distribution of contrasts in natural images was measured for 65 scenes. The results, in general agreement with other reports, show that the vast majority of contrasts in nature are very small. The efficient coding hypothesis of Laughlin was examined by comparing the average contrast/response curves of bipolar cells with the cumulative probability distribution of contrasts in natural images. Efficient coding was found at 20 cd/m2 but at lower levels of light adaptation, the contrast/response curves were much too shallow. Further experiments show that two fundamental physiological factors-light adaptation and the nonlinear transfer across the cone-bipolar synapse are essential for the emergence of efficient contrast coding. For both land- and aquatic-based animals, the extent and symmetry of the dynamic range of the contrast/response curves of both classes of bipolar cells varied greatly from cell to cell. This apparent substrate for distributed encoding is established at the bipolar cell level, since it is not found in cones. As a result, the dynamic range of the bipolar cell population brackets the distribution of contrasts found in natural images.

Retinal bipolar cells: contrast encoding for sinusoidal modulation and steps of luminance contrast.

Contrast encoding for sinusoidal modulations of luminance contrast was investigated by intracellular recording in the intact salamander retina. In what appears to be the first study of this kind for vertebrate bipolar cells, responses of the central receptive-field mechanism of cone-driven cells to modulation of 3 Hz were analyzed quantitatively via both signal averaging and a Fast Fourier Transform (FFT) while the retina was light adapted to 20 cd/m2. Depolarizing and hyperpolarizing bipolar cells showed very similar encoding. Both responded with sinusoidal waveforms whose amplitude varied linearly with modulation depths ranging up to 7-8%. The slope of the modulation/response curve was very steep in this range. Thus, the contrast gain was high, reaching values of 6-7, and the half-maximal response was achieved at modulations of 9% or less. At modulations above approximately 15%, the responses typically showed strong compressive nonlinearity and the waveform was increasingly distorted. At maximum modulation, the higher harmonics of the FFT constituted about 30% of the amplitude of the fundamental. Measurements were also made for cones and horizontal cells. Both cell types showed predominantly linear responses and low contrast gain, in marked contrast to bipolar cells. These results suggest that the high contrast gain and strong nonlinearity of bipolar cells largely arise postsynaptic to cone transmitter release. Further experiments were performed to compare responses to contrast steps versus those to sinusoidal modulation. In the linear range, we show that the contrast gains of cones and horizontal cells are low and virtually identical for both steps and sinusoidal modulations. In bipolar cells, on the other hand, the contrast gain is about two times greater for steps than that for the 3-Hz sine waves. These results suggest that mechanisms intrinsic to bipolar cells act like a high-pass filter with a short time constant to selectively emphasize contrast transients over slower changes in contrast.