Positive and negative selection shape the human naive B cell repertoire.

Although negative selection of developing B cells in the periphery is well described, yet poorly understood, evidence of naive B cell positive selection remains elusive. Using 2 humanized mouse models, we demonstrate that there was strong skewing of the expressed immunoglobulin repertoire upon transit into the peripheral naive B cell pool. This positive selection of expanded naive B cells in humanized mice resembled that observed in healthy human donors and was independent of autologous thymic tissue. In contrast, negative selection of autoreactive B cells required thymus-derived Tregs and MHC class II-restricted self-antigen presentation by B cells. Indeed, both defective MHC class II expression on B cells of patients with rare bare lymphocyte syndrome and prevention of self-antigen presentation via HLA-DM inhibition in humanized mice resulted in the production of autoreactive naive B cells. These latter observations suggest that Tregs repressed autoreactive naive B cells continuously produced by the bone marrow. Thus, a model emerged, in which both positive and negative selection shaped the human naive B cell repertoire and that each process was mediated by fundamentally different molecular and cellular mechanisms.

Autoreactivity in naïve human fetal B cells is associated with commensal bacteria recognition.

Restricted V(D)J recombination during fetal development was postulated to limit antibody repertoire breadth and prevent autoimmunity. However, newborn serum contains abundant autoantibodies, suggesting that B cell tolerance during gestation is not yet fully established. To investigate this apparent paradox, we evaluated the reactivities of more than 450 antibodies cloned from single B cells from human fetal liver, bone marrow, and spleen. We found that incomplete B cell tolerance in early human fetal life favored the accumulation of polyreactive B cells that bound both apoptotic cells and commensal bacteria from healthy adults. Thus, the restricted fetal preimmune repertoire contains potentially beneficial self-reactive innate-like B cell specificities that may facilitate the removal of apoptotic cells during development and shape gut microbiota assembly after birth.

Consensus-based classification system for intra-operative management of labral tears during hip arthroscopy-aggregate recommendations from high-volume hip preservation surgeons.

The purpose of this study was to survey high-volume hip preservation surgeons regarding their perspectives on intra-operative management of labral tears to improve decision-making and produce an effective classification system. A cross-sectional survey of high-volume hip preservation surgeons was conducted in person and anonymously, using a questionnaire that is repeated for indications of labral debridement, repair and reconstruction given the torn labra are stable, unstable, viable or non-viable. Twenty-six high-volume arthroscopic hip surgeons participated in this survey. Provided the labrum was viable (torn tissue that is likely to heal) and stable, labral debridement would be performed by 76.92% of respondents for patients >40 years of age and by >84% of respondents for stable intra-substance labral tears in patients without dysplasia. If the labrum was viable but unstable, labral repair would be performed by >80% of respondents for patients ≤40 years of age and > 80% of respondents if the labral size was >3 mm and located anteriorly. Presence of calcified labra or the Os acetabuli mattered while deciding whether to repair a labrum. In non-viable (torn tissue that is unlikely to heal) and unstable labra, labral reconstruction would be performed by 84.62% of respondents if labral size was <3 mm. The majority of respondents would reconstruct calcified and non-viable, unstable labra that no longer maintained a suction seal. Surgeons performing arthroscopic hip labral treatment may utilize this comprehensive classification system, which takes into consideration patient age, labral characteristics (viability and stability) and bony morphology of the hip joint. When choosing between labral debridement, repair or reconstruction, consensus recommendations from high-volume hip preservation surgeons can enhance decision-making.

Impaired ATM activation in B cells is associated with bone resorption in rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) may display atypical CD21-/lo B cells in their blood, but the implication of this observation remains unclear. We report here that the group of patients with RA and elevated frequencies of CD21-/lo B cells shows decreased ataxia telangiectasia-mutated (ATM) expression and activation in B cells compared with other patients with RA and healthy donor controls. In agreement with ATM involvement in the regulation of V(D)J recombination, patients with RA who show defective ATM function displayed a skewed B cell receptor (BCR) Igκ repertoire, which resembled that of patients with ataxia telangiectasia (AT). This repertoire was characterized by increased Jκ1 and decreased upstream Vκ gene segment usage, suggesting improper secondary recombination processes and selection. In addition, altered ATM function in B cells was associated with decreased osteoprotegerin and increased receptor activator of nuclear factor κB ligand (RANKL) production. These changes favor bone loss and correlated with a higher prevalence of erosive disease in patients with RA who show impaired ATM function. Using a humanized mouse model, we also show that ATM inhibition in vivo induces an altered Igκ repertoire and RANKL production by immature B cells in the bone marrow, leading to decreased bone density. We conclude that dysregulated ATM function in B cells promotes bone erosion and the emergence of circulating CD21-/lo B cells, thereby contributing to RA pathophysiology.

AIRE expression controls the peripheral selection of autoreactive B cells.

Autoimmune regulator (AIRE) mutations result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome characterized by defective central T cell tolerance and the production of many autoantibodies targeting tissue-specific antigens and cytokines. By studying CD3- and AIRE-deficient patients, we found that lack of either T cells or AIRE function resulted in the peripheral accumulation of autoreactive mature naïve B cells. Proteomic arrays and Biacore affinity measurements revealed that unmutated antibodies expressed by these autoreactive naïve B cells recognized soluble molecules and cytokines including insulin, IL-17A, and IL-17F, which are AIRE-dependent thymic peripheral tissue antigens targeted by autoimmune responses in APECED. AIRE-deficient patients also displayed decreased frequencies of regulatory T cells (Tregs) that lacked common TCRß clones found instead in their conventional T cell compartment, thereby suggesting holes in the Treg TCR repertoire of these patients. Hence, AIRE-mediated T cell/Treg selection normally prevents the expansion of autoreactive naïve B cells recognizing peripheral self-antigens.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.

Decreasing the Cerebral Edema Associated with Traumatic Intracerebral Hemorrhages: Use of a Minimally Invasive Technique.

Traumatic brain injury (TBI) is a major public health problem worldwide that affects all age groups. In the United States alone, there are approximately 50,000 deaths from severe traumatic brain injuries each year. In most studies, about 40 % of severe TBI have associated traumatic intracerebral hemorrhages (tICHs). The surgical treatment of tICH is debated largely because of its invasive nature, particularly in reaching deep tICHs. tICHs have a clear contribution to mass effect and exacerbate cerebral edema and ICP because of the break-down products of hemorrhage. We introduce a modification of the Mi SPACE technique (Minimally Invasive Subcortical Parafascicular Transsulcal Access for Clot Evacuation) that is applicable to tICH. In brief, this technique utilizes a trans-sulcal, stereotactic-guided technique in which a specially designed cannula is used to introduce a 13.5-mm-diameter tube into the epicenter of the tICH. We identified eight tICHs that were treated entirely or in part with the modified Mi SPACE technique during the time period from August 15, 2014 to December 15, 2014. This modified technique was readily deployed safely and efficaciously with significant removal of the tICH as demonstrated by postoperative CT scans. The removal of tICH using this minimally invasive technique may help with the control of ICP and cerebral edema.

Consensus statement from the 2014 International Microdialysis Forum.

Microdialysis enables the chemistry of the extracellular interstitial space to be monitored. Use of this technique in patients with acute brain injury has increased our understanding of the pathophysiology of several acute neurological disorders. In 2004, a consensus document on the clinical application of cerebral microdialysis was published. Since then, there have been significant advances in the clinical use of microdialysis in neurocritical care. The objective of this review is to report on the International Microdialysis Forum held in Cambridge, UK, in April 2014 and to produce a revised and updated consensus statement about its clinical use including technique, data interpretation, relationship with outcome, role in guiding therapy in neurocritical care and research applications.

Implementation of cerebral microdialysis at a community-based hospital: A 5-year retrospective analysis.

BACKGROUND: Cerebral microdialysis (MD) provides valuable information about brain metabolism under normal and pathologic conditions. The CMA 600 microdialysis analyzer received US Food and Drug Administration (FDA) approval for clinical use in the United States in 2005. Since then, cerebral MD has been increasingly utilized nationally in the multimodal monitoring of traumatic brain injury (TBI), stroke, aneurysmal subarachnoid hemorrhage, and brain tumors. We describe a 5-year, single-institutional experience using cerebral MD at a community-based hospital, Legacy Emanuel Medical Center (LEMC). Implications for the adoption and utility of MD in medical centers with limited resources are discussed. METHODS: This is a retrospective chart review and data analysis of 174 consecutive patients who had cerebral MD as part of multimodal brain monitoring. All cerebral MD catheters were placed by board-certified, attending neurosurgeons at LEMC. Clinical severity in the TBI patients was reported using initial Glasgow Coma Scale (GCS); radiologic severity was graded with the Marshall CT grading scale. Measures of the risks of MD placement included post-placement hemorrhage, cerebral infection, and dislodgement. RESULTS: Between July 2005 and July 2010, 248 cerebral MD catheters were placed in 174 patients undergoing multimodal brain monitoring. One hundred and eighty-five catheters were placed at the time of open craniotomy. None were associated with cranial infection. Patients ranged in age from 5 months to 90 years, with a mean of 49 years. The male to female ratio was 1.4:1. The underlying pathologies were: TBI (126), cerebral vascular accident (24), aneurysmal subarachnoid hemorrhage (17), and tumor (7). CONCLUSIONS: Cerebral MD was readily implemented in a community-based hospital. No cerebral hemorrhages or infections were attributed to cerebral MD. Examples of how MD may be a useful adjunct in the clinical decision making of patients with brain injuries are presented.

Pupillary reactivity as an early indicator of increased intracranial pressure: The introduction of the Neurological Pupil index.

BACKGROUND: This paper introduces the 7/5/2011al Pupil index (NPi), a sensitive measure of pupil reactivity and an early indicator of increasing intracranial pressure (ICP). This may occur in patients with severe traumatic brain injury (TBI), aneurysmal subarachnoid hemorrhage, or intracerebral hemorrhage (ICH). METHODS: 134 patients (mean age 46 years, range 18-87 years, 54 women and 80 men) in the intensive care units at eight different clinical sites were enrolled in the study. Pupillary examination was performed using a portable hand-held pupillometer. RESULTS: Patients with abnormal pupillary light reactivity had an average peak ICP of 30.5 mmHg versus 19.6 mmHg for the normal pupil reactivity population (P = 0.0014). Patients with "nonreactive pupils" had the highest peaks of ICP (mean = 33.8 mmHg, P = 0.0046). In the group of patients with abnormal pupillary reactivity, we found that the first evidence of pupil abnormality occurred, on average, 15.9 hours prior to the time of the peak of ICP. CONCLUSIONS: Automated pupillary assessment was used in patients with possible increased ICP. Using NPi, we were able to identify a trend of inverse relationship between decreasing pupil reactivity and increasing ICP. Quantitative measurement and classification of pupillary reactivity using NPi may be a useful tool in the early management of patients with causes of increased ICP.

Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly people over 60. The pathogenesis is still unclear. It has been suggested that lysosomal stress may lead to drusen formation, a biomarker of AMD. In this study, ARPE-19 cells were treated with chloroquine to inhibit lysosomal function. RESULTS: Chloroquine-treated ARPE-19 cells demonstrate a marked increase in vacuolation and dense intracellular debris. These are identified as chloroquine-dilated lysosomes and lipid bodies with LAMP-2 and LipidTOX co-localization, respectively. Dilation is an indicator of lysosomal dysfunction. Chloroquine disrupts uptake of exogenously applied rhodamine-labeled dextran by these cells. This suggests a disruption in the phagocytic pathway. The increase in LAMP protein levels, as assessed by Western blots, suggests the possible involvement in autophagy. Oxidative stress with H2O2 does not induce vacuolation or lipid accumulation. CONCLUSION: These findings suggest a possible role for lysosomes in AMD. Chloroquine treatment of RPE cells may provide insights into the cellular mechanisms underlying AMD.

Acid-sensing ion channels in acidosis-induced injury of human brain neurons.

Acidosis is a common feature of the human brain during ischemic stroke and is known to cause neuronal injury. However, the mechanism underlying acidosis-mediated injury of the human brain remains elusive. We show that a decrease in the extracellular pH evoked inward currents characteristic of acid-sensing ion channels (ASICs) and increased intracellular Ca(2+) in cultured human cortical neurons. Acid-sensing ion channels in human cortical neurons show electrophysiological and pharmacological properties distinct from those in neurons of the rodent brain. Reverse transcriptase-PCR and western blot detected a high level of the ASIC1a subunit with little or no expression of other ASIC subunits. Treatment of human cortical neurons with acidic solution induced substantial cell injury, which was attenuated by the ASIC1a blockade. Thus, functional homomeric ASIC1a channels are predominantly expressed in neurons from the human brain. Activation of these channels has an important role in acidosis-mediated injury of human brain neurons.

Cervical spine injuries.

This article describes the anatomy of the cervical spine and the most common types of fractures associated with the cervical spine. Cervical spinal cord syndromes are also reviewed because such syndromes discovered during neurologic examinations frequently provide the first clue that there is an underlying spinal cord injury. Because most associated maxillofacial and spinal injuries occur in the setting of motor vehicle accidents, it is particularly important for the maxillofacial surgeon to be cognizant of the injuries, particularly in the context of the need for facial/cranial surgery. Appropriate measures are necessary to immobilize or fixate the spine before surgery to avoid exacerbating the spinal injury.

Quantitative pupillometry, a new technology: normative data and preliminary observations in patients with acute head injury. Technical note.

The authors prospectively used a new hand-held point-and-shoot pupillometer to assess pupillary function quantitatively. Repetitive measurements were initially made in more than 300 healthy volunteers ranging in age from 1 to 87 years, providing a total of 2,432 paired (alternative right eye, left eye) measurements under varying light conditions. The authors studied 17 patients undergoing a variety of nonintracranial, nonophthalmological, endoscopic, or surgical procedures and 20 seniors in a cardiology clinic to learn more about the effects of a variety of drugs. Additionally, the authors carried out detailed studies in 26 adults with acute severe head injury in whom intracranial pressure (ICP) was continuously monitored. Finally, five patients suffering from subarachnoid hemorrhage were also studied. Quantitative pupillary measurements could be reliably replicated in the study participants. In healthy volunteers the resting pupillary aperture averaged 4.1 mm and the minimal aperture after stimulation was 2.7 mm, resulting in a 34% change in pupil size. Constriction velocity averaged 1.48 +/- 0.33 mm/second. Pupillary symmetry was striking in both healthy volunteers and patients without intracranial or uncorrected visual acuity disorders. In the 2,432 paired measurements in healthy volunteers, constriction velocity was noted to fall below 0.85 mm/second on only 33 occasions and below 0.6 mm/second on eight occasions (< one in 310 observations). In outpatients, the reduction in constriction velocity was observed when either oral or intravenous narcotic agents and diazepam analogs were administered. These effects were transient and always symmetrical. Among the 26 patients with head injuries, eight were found to have elevations of ICP above 20 mm Hg and pupillary dynamics in each of these patients remained normal. In 13 patients with a midline shift greater than 3 mm, elevations of ICP above 20 mm Hg, when present for 15 minutes, were frequently associated with a reduction in constriction velocity on the side of the mass effect to below 0.6 mm/second (51% of 156 paired observations). In five patients with diffuse brain swelling but no midline shift, a reduction in constriction velocities did not generally occur until the ICP exceeded 30 mm Hg. Changes in the percentage of reduction from the resting state following stimulation were always greater than 10%, even in patients receiving large doses of morphine and propofol in whom the ICP was lower than 20 mm Hg. Asymmetry of pupillary size greater than 0.5 mm was observed infrequently (< 1%) in healthy volunteers and was rarely seen in head-injured patients unless the ICP exceeded 20 mm Hg. Pupillometry is a reliable technology capable of providing repetitive data on quantitative pupillary function in states of health and disease.