A methodology for specific disruption of microtubule polymerization into dendritic spines.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

A Methodology for Specific Disruption of Microtubules in Dendritic Spines.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

Identification of enhanced IFN-γ signaling in polyarticular juvenile idiopathic arthritis with mass cytometry.

Polyarticular juvenile idiopathic arthritis (JIA) is among the most challenging of the JIA subtypes to treat. Even with current biologic therapies, the disease remains difficult to control in a substantial subset of patients, highlighting the need for new therapies. The aim of this study was to use the high dimensionality afforded by mass cytometry with phospho-specific antibodies to delineate signaling abnormalities in immune cells from treatment-naive polyarticular JIA patients. Peripheral blood mononuclear cells were isolated from 17 treatment-naive polyarticular JIA patients, 10 of the patients after achieving clinical remission, and 19 healthy controls. Samples were stimulated for 15 minutes with IL-6 or IFN-γ and analyzed by mass cytometry. Following IFN-γ stimulation, increased STAT1 and/or STAT3 phosphorylation was observed in subsets of CD4 T cells and classical monocytes from treatment-naive patients. The enhanced IFN-γ signaling was associated with increased expression of JAK1 and SOCS1 in CD4 T cells. Furthermore, substantial heterogeneity in surface marker expression was observed among the subsets of CD4 T cells and classical monocytes with increased IFN-γ responsiveness. The identification of enhanced IFN-γ signaling in CD4 T cells and classical monocytes from treatment-naive polyarticular JIA patients provides mechanistic support for investigations into therapies that attenuate IFN-γ signaling in this disease.

An Exploratory Study of the Knowledge of Personal Safety Skills Among Children with Developmental Disabilities and Their Parents.

BACKGROUND: This study assessed the knowledge of personal safety skills among children with developmental disabilities and their parents' perceptions of children's knowledge. METHOD: This exploratory study examined the mental health records of 37 children with developmental disabilities referred for an abuse risk reduction group in a community mental health setting. Qualitative analysis of children's responses to questions about personal safety skills (knowledge related to physical development and personal safety, an appropriate and inappropriate touch and safety skills to respond to an inappropriate touch) indicated participants' varied and inconsistent levels of knowledge. RESULTS: Consistent with the literature, the results indicate risk factors for sexual abuse among children with developmental disabilities, including children's difficulty distinguishing between an appropriate and inappropriate touch and the lack of knowledge regarding appropriate venues for disclosing an inappropriate touch. Among parents, a lack of certainty regarding their children's knowledge and the ability to keep themselves safe was identified. CONCLUSION: Results support the need for education about personal safety for children with developmental disabilities and their families.

Manifestations of inflammatory bowel disease in patients of Haitian and Cape Verdean descent.

OBJECTIVE: Several studies have reported unique ethnic phenotypes of inflammatory bowel disease (IBD). An appreciation of disease manifestations in different populations may improve clinical outcomes. There are no studies examining IBD in patients of Haitian or Cape Verdean descent. We sought to define the IBD phenotype in these populations. MATERIALS AND METHODS: This was a retrospective review comparing Haitian and Cape Verdean immigrant IBD patients to Caucasians, all receiving care at Boston Medical Center in Boston, Massachusetts, USA. The following variables were analyzed: family history, smoking history, vaccinations/cancer screening, age of diagnosis, disease duration, disease location, medication use, and complications. RESULTS: Thirty-one Haitians and 21 Cape Verdeans were matched to Caucasian controls. Haitians (mean age 42 years) and Cape Verdeans (mean age 47 years) with Crohn's disease were diagnosed with IBD later than Caucasians (mean age 31 years, p = 0.04 and 0.02, respectively). Haitians with Crohn's were less likely to have a history of tobacco use compared to Caucasians (13% vs. 51%, p = 0.02). Cape Verdeans with Crohn's were less likely to have perianal involvement (0% vs. 50%, p = 0.01). Haitians with IBD were less likely to have ever used glucocorticoids (48% vs. 76%, p = 0.02). There was no difference in vaccination rates, cancer screening, or disease complications. CONCLUSIONS: This study demonstrates differences in IBD presentation and disease course among Haitians and Cape Verdeans. Our results suggest a more mild disease in these ethnic groups. Future studies are needed to identify the influence of environmental factors.

Influenza and Pneumococcal Vaccination Rates in Patients With Inflammatory Bowel Disease.

Patients with inflammatory bowel disease (IBD) are at an increased risk for vaccine-preventable illnesses, such as pneumococcal pneumonia and influenza. We hypothesized that a patient-directed educational program would increase vaccination rates of patients with IBD. We developed a written educational form that was given to all patients over a 15-month period. The form included information about the importance of vaccination and asked patients about their vaccination status. If patients indicated that they were not vaccinated, they were offered a vaccination at the time of their visit. For influenza, the vaccination rates during 3 seasons were compared. For pneumococcal pneumonia, the vaccination rates during a 6-month period before the introduction of the educational program and the rates during the 15-month period after implementation of the intervention were compared. Our form increased the percentage of patients who reported having an influenza vaccination (23% vs 47%; P<.001) and the percentage of patients who reported having a pneumococcal pneumonia vaccination (21% vs 32%; P<.001). We concluded that a simple written educational form designed to assess vaccination status and enable providers to offer same-day influenza and pneumococcal pneumonia vaccinations resulted in a significant increase in influenza and pneumococcal pneumonia vaccination rates among patients in an IBD specialty clinic.

CD8(+)IL-17(+) T Cells Mediate Neutrophilic Airway Obliteration in T-bet-Deficient Mouse Lung Allograft Recipients.

Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.

Effect of propofol anesthesia on force application during colonoscopy.

BACKGROUND: Sedation is frequently used during colonoscopy to control patient discomfort and pain. Propofol is associated with a deeper level of sedation than is a combination of a narcotic and sedative hypnotic and, therefore, may be associated with an increase in force applied to the colonoscope to advance and withdraw the instrument. OBJECTIVE: To compare force application to the colonoscope insertion tube during propofol anesthesia and moderate sedation. DESIGN: An observational cohort study of 13 expert and 12 trainee endoscopists performing colonoscopy in 114 patients. Forces were measured by using the colonoscopy force monitor, which is a wireless, handheld device that attaches to the insertion tube of the colonoscope. SETTING: Community ambulatory surgery center and academic gastroenterology training programs. PATIENTS: Patients undergoing routine screening or diagnostic colonoscopy with complete segment force recordings. MAIN OUTCOME MEASUREMENTS: Axial and radial forces and examination time. RESULTS: Axial and radial forces increase and examination time decreases significantly when propofol is used as the method of anesthesia. LIMITATIONS: Small study, observational design, nonrandomized distribution of sedation type and experience level, different instrument type and effect of prototype device on insertion tube manipulation. CONCLUSIONS: Propofol sedation is associated with a decrease in examination time and an increase in axial and radial forces used to advance the colonoscope.

Effects of reinforcer magnitude on reinforced behavioral variability.

Eight pigeons were exposed to a two-component multiple schedule. In each component, four-peck sequences across left and right keys were reinforced according to a variability threshold contingency. In one condition, only infrequently occurring response sequences were reinforced in each component, thereby generating highly variable sequences. In a separate condition, when the variability threshold contingency was lenient in each component, sequences were much less variable. In each condition, reinforcer magnitude was manipulated across components, and the larger reinforcer magnitude produced less variability than the smaller reinforcer magnitude. These results suggest that larger reinforcers hinder the reinforcement of behavioral variability. The results are interpretable in terms of the larger reinforcer inducing a greater level of behavioral repetition, particularly as the time to reinforcement was approached. This effect may have implications for reinforcing behavioral variability in humans.

Prevention of airway allograft tolerance by polyinosinic:polycytidylic acid requires type I interferon responsiveness for mouse airway obliteration.

BACKGROUND: Respiratory RNA viruses are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients (LTRS); however, the immune mechanisms that regulate airway obliteration remain incompletely understood. METHODS: Using the mouse heterotopic tracheal transplant model of obliterative airway disease (OAD), we studied the role of double-stranded (ds)RNA using polyinosinic:polycytidylic acid (poly[I:C]), a synthetic analog of viral dsRNA, in abrogating airway allograft tolerance established with donor-specific transfusion (DST) and anti-CD154 monoclonal antibody therapy. RESULTS: Wild-type (WT) B6 recipients of accepted BALB/c airway grafts demonstrated significantly reduced intragraft CD8+ T cells, with markedly impaired allospecific interferon (INF)-γ and tumor necrosis factor-α secretion, uncoupled from an activated phenotype, and evidence of proliferation. Administration of poly(I:C) to DST/anti-CD154-treated recipients restored OAD pathology and CD8+ alloeffector responses to levels observed in untreated mice. However, B6 type I IFN receptor-deficient (IFN-αßR(-/-)) recipients were resistant to the abrogation of tolerance mediated by poly(I:C) and did not develop CD8+ alloeffector responses or OAD. Further, adoptive transfers of WT CD8+ T cells or CD11c+ dendritic cells alone into B6 IFNαßR(-/-) recipients treated with poly(I:C) and DST/anti-CD154 were incapable of abrogating airway graft tolerance. CONCLUSIONS: Together, these data indicate abrogation of DST/anti-CD154-induced airway allograft tolerance via dsRNA requires type-I IFN responsiveness for mouse airway obliteration.

Segmental increases in force application during colonoscope insertion: quantitative analysis using force monitoring technology.

BACKGROUND: Colonoscopy is a frequently performed procedure that requires extensive training and a high skill level. OBJECTIVE: Quantification of forces applied to the external portion of the colonoscope insertion tube during the insertion phase of colonoscopy. DESIGN: Observational cohort study of 7 expert and 9 trainee endoscopists for analysis of colonic segment force application in 49 patients. Forces were measured by using the colonoscopy force monitor, which is a wireless, handheld device that attaches to the insertion tube of the colonoscope. SETTING: Academic gastroenterology training programs. PATIENTS: Patients undergoing routine screening or diagnostic colonoscopy with complete segment force recordings. MAIN OUTCOME MEASUREMENTS: Axial and radial force and examination time. RESULTS: Both axial and radial force increased significantly as the colonoscope was advanced from the rectum to the cecum. Analysis of variance demonstrated highly significant operator-independent differences between segments of the colon (zones) in all axial and radial forces except average torque. Expert and trainee endoscopists differed only in the magnitude of counterclockwise force, average push/pull force rate used, and examination time. LIMITATIONS: Small study, observational design, effect of prototype device on insertion tube manipulation. CONCLUSION: Axial and radial forces used to advance the colonoscope increase through the segments of the colon and are operator independent.

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation.

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G(0) phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G(0) phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCF(FBXL2) in lymphoproliferative malignancies.

Colonoscopy surveillance after polypectomy may be extended beyond five years.

OBJECTIVES: Colonoscopy surveillance interval data longer than 5 years are limited. We examined adenoma yield to identify factors that predict appropriate intervals for postpolypectomy surveillance greater than 5 years, including risk of advanced adenoma recurrence. METHODS: We identified patients with and without adenomas on an index colonoscopy who returned at 5 to 10 years for a follow-up colonoscopy. Multivariate logistic regression was used to identify variables that predict finding an adenoma on follow-up colonoscopy. RESULTS: Three hundred ninety-nine patients were identified with a follow-up colonoscopy at an interval of >5 years. Irrespective of surveillance interval, adenoma incidence occurred in 116 patients (29.1%) with 25 (6%) having advanced adenomas. Patients with nonadvanced adenomas on index colonoscopy had a similar risk of advanced adenoma on follow-up colonoscopy at 5 years versus 6 to 10 years, 5% versus 6.2% (P=0.39). The risk of advanced adenoma at 5 and 6 to 10 years in patients with a negative index colonoscopy was 7% versus 3.6% (P=0.15). Patients with an advanced adenoma at index colonoscopy had the highest rate of advanced adenoma detection at 5 years at 26%. Proximal polyp location (odds ratio 12.4, confidence interval 2.7-56.7) predicted advanced adenoma occurrence at 5 years. CONCLUSIONS: Postpolypectomy colonoscopy intervals can be extended beyond 5 years in patients with nonadvanced adenomas. Our findings also support a rescreening interval of 5 to 10 years in patients with a negative index colonoscopy. Patients with an index advanced adenoma are at highest risk for recurrent advanced adenoma and should have repeat colonoscopy before a 5 years interval.