The Influence of COVID-19 on Patient Mobilization and Injury Attributes in the ICU: A Retrospective Analysis of a Level II Trauma Center.

The objectives of this study were to determine the effect of COVID-19 on physical therapy (PT) mobilization of trauma patients and to determine if mobilization affected patient course in the ICU. This retrospective study included patients who were admitted to the ICU of a level II trauma center. The patients were divided into two groups, i.e., those admitted before (n = 378) and after (n = 499) 1 April 2020 when Georgia's COVID-19 shelter-in-place order was mandated. The two groups were contrasted on nominal and ratio variables using Chi-square and Student's t-tests. A secondary analysis focused specifically on the after-COVID patients examined the extent to which mobilization (n = 328) or lack of mobilization (n = 171) influenced ICU outcomes (e.g., mortality, readmission). The two groups were contrasted on nominal and ratio variables using Chi-square and Student's t-tests. The after-COVID patients had higher injury severity as a greater proportion was classified as severely injured (i.e., >15 on Injury Severity Score) compared to the before-COVID patients. After-COVID patients also had a greater cumulative number of comorbidities and experienced greater complications in the ICU. Despite this, there was no difference between patients in receiving a PT consultation or days to mobilization. Within the after-COVID cohort, those who were mobilized were older, had greater Glasgow Coma Scale scores, had longer total hospital days, and had a lesser mortality rate, and a higher proportion were female. Despite shifting patient injury attributes post-COVID-19, a communicable disease, mobilization care remained consistent and effective.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) surface contamination in staff common areas and impact on healthcare worker infection: Prospective surveillance during the coronavirus disease 2019 (COVID-19) pandemic.

We prospectively surveyed SARS-CoV-2 RNA contamination in staff common areas within an acute-care hospital. An increasing prevalence of surface contamination was detected over time. Adjusting for patient census or community incidence of coronavirus disease 2019 (COVID-19), the proportion of contaminated surfaces did not predict healthcare worker COVID-19 infection on study units.

SARS-CoV-2 RNA persists on surfaces following terminal disinfection of COVID-19 hospital isolation rooms.

We evaluated the effect of terminal cleaning on SARS-CoV-2 RNA contamination of COVID-19 isolation rooms in an acute care hospital. SARS-CoV-2 RNA was detected on 32.1% of room surfaces after cleaning; the odds of contamination increased with month. The prevalence of elevated high-touch surface contamination was lower in terminally cleaned rooms than patient-occupied rooms.

Healthcare microenvironments define multidrug-resistant organism persistence.

BACKGROUND: Multidrug-resistant organisms (MDROs) colonizing the healthcare environment have been shown to contribute to risk for healthcare-associated infections (HAIs), with adverse effects on patient morbidity and mortality. We sought to determine how bacterial contamination and persistent MDRO colonization of the healthcare environment are related to the position of patients and wastewater sites. METHODS: We performed a prospective cohort study, enrolling 51 hospital rooms at the time of admitting a patient with an eligible MDRO in the prior 30 days. We performed systematic sampling and MDRO culture of rooms, as well as 16S rRNA sequencing to define the environmental microbiome in a subset of samples. RESULTS: The probability of detecting resistant gram-negative organisms, including Enterobacterales, Acinetobacter spp, and Pseudomonas spp, increased with distance from the patient. In contrast, Clostridioides difficile and methicillin-resistant Staphylococcus aureus were more likely to be detected close to the patient. Resistant Pseudomonas spp and S. aureus were enriched in these hot spots despite broad deposition of 16S rRNA gene sequences assigned to the same genera, suggesting modifiable factors that permit the persistence of these MDROs. CONCLUSIONS: MDRO hot spots can be defined by distance from the patient and from wastewater reservoirs. Evaluating how MDROs are enriched relative to bacterial DNA deposition helps to identify healthcare micro-environments and suggests how targeted environmental cleaning or design approaches could prevent MDRO persistence and reduce infection risk.

Spatial and temporal effects on severe acute respiratory coronavirus virus 2 (SARS-CoV-2) contamination of the healthcare environment.

BACKGROUND: The spatial and temporal extent of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) environmental contamination has not been precisely defined. We sought to elucidate contamination of different surface types and how contamination changes over time. METHODS: We sampled surfaces longitudinally within COVID-19 patient rooms, performed quantitative RT-PCR for the detection of SARS-CoV-2 RNA, and modeled distance, time, and severity of illness on the probability of detecting SARS-CoV-2 using a mixed-effects binomial model. RESULTS: The probability of detecting SARS-CoV-2 RNA in a patient room did not vary with distance. However, we found that surface type predicted probability of detection, with floors and high-touch surfaces having the highest probability of detection: floors (odds ratio [OR], 67.8; 95% credible interval [CrI], 36.3-131) and high-touch elevated surfaces (OR, 7.39; 95% CrI, 4.31-13.1). Increased surface contamination was observed in room where patients required high-flow oxygen, positive airway pressure, or mechanical ventilation (OR, 1.6; 95% CrI, 1.03-2.53). The probability of elevated surface contamination decayed with prolonged hospitalization, but the probability of floor detection increased with the duration of the local pandemic wave. CONCLUSIONS: Distance from a patient's bed did not predict SARS-CoV-2 RNA deposition in patient rooms, but surface type, severity of illness, and time from local pandemic wave predicted surface deposition.

Gut microbiota features associated with Clostridioides difficile colonization in dairy calves.

Diarrheal disease, a major cause of morbidity and mortality in dairy calves, is strongly associated with the health and composition of the gut microbiota. Clostridioides difficile is an opportunistic pathogen that proliferates and can produce enterotoxins when the host experiences gut dysbiosis. However, even asymptomatic colonization with C. difficile can be associated with differing degrees of microbiota disruption in a range of species, including people, swine, and dogs. Little is known about the interaction between C. difficile and the gut microbiota in dairy calves. In this study, we sought to define microbial features associated with C. difficile colonization in pre-weaned dairy calves less than 2 weeks of age. We characterized the fecal microbiota of 80 calves from 23 different farms using 16S rRNA sequencing and compared the microbiota of C. difficile-positive (n = 24) and C. difficile-negative calves (n = 56). Farm appeared to be the greatest source of variability in the gut microbiota. When controlling for calf age, diet, and farm location, there was no significant difference in Shannon alpha diversity (P = 0.50) or in weighted UniFrac beta diversity (P = 0.19) between C. difficile-positive and-negative calves. However, there was a significant difference in beta diversity as assessed using Bray-Curtiss diversity (P = 0.0077), and C. difficile-positive calves had significantly increased levels of Ruminococcus (gnavus group) (Adj. P = 0.052), Lachnoclostridium (Adj. P = 0.060), Butyricicoccus (Adj. P = 0.060), and Clostridium sensu stricto 2 compared to C. difficile-negative calves. Additionally, C. difficile-positive calves had fewer microbial co-occurrences than C. difficile-negative calves, indicating reduced bacterial synergies. Thus, while C. difficile colonization alone is not associated with dysbiosis and is therefore unlikely to result in an increased likelihood of diarrhea in dairy calves, it may be associated with a more disrupted microbiota.

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration.

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that Il22 expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.

Quantifying the Impact of Nasopharyngeal Specimen Quality on Severe Acute Respiratory Syndrome Coronavirus 2 Test Performance.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with coronavirus disease 2019 (COVID-19) and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. METHODS: We performed amplification of a human gene target (ß-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1282 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by late Ct values for the human gene target ß-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and ß-actin Ct. RESULTS: Low-quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio, 0.607 [95% credible interval {CrI}, .487-.753]). We observed a positive linear relationship between SARS-CoV-2 and ß-actin Ct values (slope, 0.181 [95% CrI, .097-.264]), consistent with a reduction in detection of 0.181 cycles for each additional cycle of the ß-actin target. COVID-19 disease severity was not associated with ß-actin Ct values. CONCLUSIONS: Variability in NP specimen quality significantly impacts the performance of clinical SARS-CoV-2 assays, and caution should be taken when interpreting quantitative SARS-CoV-2 Ct results. If unrecognized, low-quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the successful application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c+ cells (AhRΔCD11c) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of Il1b and Marco, a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhRΔCD11c mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4- monocyte-dependent subset of macrophages as increased in AhRΔCD11c mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.

Clostridioides difficile on dairy farms and potential risk to dairy farm workers.

Clostridioides difficile causes severe colitis in people and is a significant enteric pathogen in many species of animals, including swine, horses, and potentially cattle. C. difficile is shed in feces, and transmission occurs horizontally via the fecal-oral route. Livestock has been suggested as a potential reservoir for C. difficile, and while studies have shown that swine and farm workers can be colonized with identical clones of C. difficile, the zoonotic transmission of C. difficile from livestock to people has not been definitively demonstrated. The goal of this study was to determine whether dairy calves and dairy farm workers harbored genetically similar isolates of C. difficile. First, we validated a glove juice protocol for detecting C. difficile on farm workers' hands. We then visited 23 farms and collected 1) fecal samples from 92 dairy calves, 2) hand rinsates from 38 dairy farm workers, and 3) fecal samples from five of the dairy farm workers who were willing to submit them. All samples underwent anaerobic culture and qPCR to detect C. difficile. C. difficile was detected on 15 of the farms (65.2%, 95% confidence interval (CI) 42.7%-83.6%) and in 28 calves (30.4%, 95% CI 21.2-40.9%) but in none of the hand rinsates or human fecal samples. Thus, the zoonotic transmission of C. difficile on dairy farms could not be demonstrated, and dairy farmers did not appear to be at increased risk of acquiring C. difficile via the fecal-oral route.

Effect of a Coordinated Community and Chronic Care Model Team Intervention vs Usual Care on Systolic Blood Pressure in Patients With Stroke or Transient Ischemic Attack: The SUCCEED Randomized Clinical Trial.

Importance: Few stroke survivors meet recommended cardiovascular goals, particularly among racial/ethnic minority populations, such as Black or Hispanic individuals, or socioeconomically disadvantaged populations. Objective: To determine if a chronic care model-based, community health worker (CHW), advanced practice clinician (APC; including nurse practitioners or physician assistants), and physician team intervention improves risk factor control after stroke in a safety-net setting (ie, health care setting where all individuals receive care, regardless of health insurance status or ability to pay). Design, Setting, and Participants: This randomized clinical trial included participants recruited from 5 hospitals serving low-income populations in Los Angeles County, California, as part of the Secondary Stroke Prevention by Uniting Community and Chronic Care Model Teams Early to End Disparities (SUCCEED) clinical trial. Inclusion criteria were age 40 years or older; experience of ischemic or hemorrhagic stroke or transient ischemic attack (TIA) no more than 90 days prior; systolic blood pressure (BP) of 130 mm Hg or greater or 120 to 130 mm Hg with history of hypertension or using hypertensive medications; and English or Spanish language proficiency. The exclusion criterion was inability to consent. Among 887 individuals screened for eligibility, 542 individuals were eligible, and 487 individuals were enrolled and randomized, stratified by stroke type (ischemic or TIA vs hemorrhagic), language (English vs Spanish), and site to usual care vs intervention in a 1:1 fashion. The study was conducted from February 2014 to September 2018, and data were analyzed from October 2018 to November 2020. Interventions: Participants randomized to intervention were offered a multimodal coordinated care intervention, including hypothesized core components (ie, ≥3 APC clinic visits, ≥3 CHW home visits, and Chronic Disease Self-Management Program workshops), and additional telephone visits, protocol-driven risk factor management, culturally and linguistically tailored education materials, and self-management tools. Participants randomized to the control group received usual care, which varied by site but frequently included a free BP monitor, self-management tools, and linguistically tailored information materials. Main Outcomes and Measures: The primary outcome was change in systolic BP at 12 months. Secondary outcomes were non-high density lipoprotein cholesterol, hemoglobin A1c, and C-reactive protein (CRP) levels, body mass index, antithrombotic adherence, physical activity level, diet, and smoking status at 12 months. Potential mediators assessed included access to care, health and stroke literacy, self-efficacy, perceptions of care, and BP monitor use. Results: Among 487 participants included, the mean (SD) age was 57.1 (8.9) years; 317 (65.1%) were men, and 347 participants (71.3%) were Hispanic, 87 participants (18.3%) were Black, and 30 participants (6.3%) were Asian. A total of 246 participants were randomized to usual care, and 241 participants were randomized to the intervention. Mean (SD) systolic BP improved from 143 (17) mm Hg at baseline to 133 (20) mm Hg at 12 months in the intervention group and from 146 (19) mm Hg at baseline to 137 (22) mm Hg at 12 months in the usual care group, with no significant differences in the change between groups. Compared with the control group, participants in the intervention group had greater improvements in self-reported salt intake (difference, 15.4 [95% CI, 4.4 to 26.0]; P = .004) and serum CRP level (difference in log CRP, -0.4 [95% CI, -0.7 to -0.1] mg/dL; P = .003); there were no differences in other secondary outcomes. Although 216 participants (89.6%) in the intervention group received some of the 3 core components, only 35 participants (14.5%) received the intended full dose. Conclusions and Relevance: This randomized clinical trial of a complex multilevel, multimodal intervention did not find vascular risk factor improvements beyond that of usual care; however, further studies may consider testing the SUCCEED intervention with modifications to enhance implementation and participant engagement. Trial Registration: ClinicalTrials.gov Identifier: NCT01763203.

Impact of Nasopharyngeal Specimen Quality on SARS-CoV-2 Test Sensitivity.

BACKGROUND: The SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) cycle of threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with COVID-19 and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. METHODS: We performed amplification of a human gene target (ß-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1311 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by high Ct values for the human gene target ß-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and ß-actin Ct. RESULTS: Low quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio 0.654, 95%CI 0.523 to 0.802). We observed a positive linear relationship between SARS-CoV-2 and ß-actin Ct values (slope 0.169, 95%CI 0.092 to 0.247). COVID-19 disease severity was not associated with ß-actin Ct values. CONCLUSIONS: Variability in NP specimen quality accounts for significant differences in the sensitivity of clinical SARS-CoV-2 assays. If unrecognized, low quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

Factors Associated With Participation in the Chronic Disease Self-Management Program: Findings From the SUCCEED Trial.

BACKGROUND AND PURPOSE: Self-management programs may improve quality of life and self-efficacy for stroke survivors, but participation is low. In a randomized controlled trial of a complex, multidisciplinary, team-based secondary stroke prevention intervention, we offered participants Chronic Disease Self-Management Program (CDSMP) workshops in addition to clinic visits and home visits. To enhance participation, workshops were facilitated by community health workers who were culturally and linguistically concordant with most participants and scheduled CDSMP sessions at convenient venues and times. Over time, we implemented additional strategies such as free transportation and financial incentives. In this study, we aimed to determine factors associated with CDSMP participation and attendance. METHODS: From 2014 to 2018, 18 CDSMP workshop series were offered to 241 English and Spanish-speaking individuals (age ≥40 years) with recent stroke or transient ischemic attack. Zero-inflated Poisson regression was used to identify factors associated with participation and attendance (ie, number of sessions attended) in CDSMP. Missing values were imputed using multiple imputation methods. RESULTS: Nearly one-third (29%) of intervention subjects participated in CDSMP. Moderate disability and more clinic/home visits were associated with participation. Participants with higher numbers of clinic and home visits (incidence rate ratio [IRR], 1.06 [95% CI, 1.01-1.12]), severe (IRR, 2.34 [95% CI, 1.65-3.31]), and moderately severe disability (IRR, 1.55 [95% CI, 1.07-2.23]), and who enrolled later in the study (IRR, 1.12 [95% CI, 1.08-1.16]) attended more sessions. Individuals with higher chaos scores attended fewer sessions (IRR, 0.97 [95% CI, 0.95-0.99]). CONCLUSIONS: Less than one-third of subjects enrolled in the SUCCEED (Secondary Stroke Prevention by Uniting Community and Chronic Care Model Teams Early to End Disparities) intervention participated in CDSMP; however, participation improved as transportation and financial barriers were addressed. Strategies to address social determinants of health contributing to chaos and engage individuals in healthcare may facilitate attendance. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01763203.

Discovery of a Potent and Selective Steroidal Glucocorticoid Receptor Antagonist (ORIC-101).

The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

Randomized controlled trial of a coordinated care intervention to improve risk factor control after stroke or transient ischemic attack in the safety net: Secondary stroke prevention by Uniting Community and Chronic care model teams Early to End Disparities (SUCCEED).

BACKGROUND: Recurrent strokes are preventable through awareness and control of risk factors such as hypertension, and through lifestyle changes such as healthier diets, greater physical activity, and smoking cessation. However, vascular risk factor control is frequently poor among stroke survivors, particularly among socio-economically disadvantaged blacks, Latinos and other people of color. The Chronic Care Model (CCM) is an effective framework for multi-component interventions aimed at improving care processes and outcomes for individuals with chronic disease. In addition, community health workers (CHWs) have played an integral role in reducing health disparities; however, their effectiveness in reducing vascular risk among stroke survivors remains unknown. Our objectives are to develop, test, and assess the economic value of a CCM-based intervention using an Advanced Practice Clinician (APC)-CHW team to improve risk factor control after stroke in an under-resourced, racially/ethnically diverse population. METHODS/DESIGN: In this single-blind randomized controlled trial, 516 adults (≥40 years) with an ischemic stroke, transient ischemic attack or intracerebral hemorrhage within the prior 90 days are being enrolled at five sites within the Los Angeles County safety-net setting and randomized 1:1 to intervention vs usual care. Participants are excluded if they do not speak English, Spanish, Cantonese, Mandarin, or Korean or if they are unable to consent. The intervention includes a minimum of three clinic visits in the healthcare setting, three home visits, and Chronic Disease Self-Management Program group workshops in community venues. The primary outcome is blood pressure (BP) control (systolic BP <130 mmHg) at 1 year. Secondary outcomes include: (1) mean change in systolic BP; (2) control of other vascular risk factors including lipids and hemoglobin A1c, (3) inflammation (C reactive protein [CRP]), (4) medication adherence, (5) lifestyle factors (smoking, diet, and physical activity), (6) estimated relative reduction in risk for recurrent stroke or myocardial infarction (MI), and (7) cost-effectiveness of the intervention versus usual care. DISCUSSION: If this multi-component interdisciplinary intervention is shown to be effective in improving risk factor control after stroke, it may serve as a model that can be used internationally to reduce race/ethnic and socioeconomic disparities in stroke in resource-constrained settings. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01763203 .

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Neuronal expression of the Ccm2 gene in a new mouse model of cerebral cavernous malformations.

Cerebral cavernous malformations are vascular defects of the central nervous system consisting of clusters of dilated vessels that are subject to frequent hemorrhaging. The genes mutated in three forms of autosomal dominant cerebral cavernous malformations have been cloned, but it remains unclear which cell type is ultimately responsible for the lesion. In this article we describe mice with a gene trap insertion in the Ccm2 gene. Consistent with the human phenotype, heterozygous animals develop cerebral vascular malformations, although penetrance is low. Beta-galactosidase activity in heterozygous brain and in situ hybridization in wild-type brain revealed Ccm2 expression in neurons and choroid plexus but not in vascular endothelium of small vessels in the brain. The expression pattern of Ccm2 is similar to that of the Ccm1 gene and its interacting protein ICAP1 (Itgb1bp1). These data suggest that cerebral cavernous malformations arise as a result of defects in the neural parenchyma surrounding the vascular endothelial cells in the brain.

Mutations in a gene encoding a novel protein containing a phosphotyrosine-binding domain cause type 2 cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in hemorrhagic stroke, seizures, recurrent headaches, and focal neurologic deficits. Mutations in the gene KRIT1 are responsible for type 1 CCM (CCM1). We report that a novel gene, MGC4607, exhibits eight different mutations in nine families with type 2 CCM (CCM2). MGC4607, similar to the KRIT1 binding partner ICAP1alpha, encodes a protein with a phosphotyrosine-binding domain. This protein may be part of the complex pathway of integrin signaling that, when perturbed, causes abnormal vascular morphogenesis in the brain, leading to CCM formation.