The presence or absence of midsagittal tissue bridges and walking: a retrospective cohort study in spinal cord injury.

STUDY DESIGN: Cohort study. Retrospective analysis of T2-weighted magnetic resonance images (MRIs) and clinical documentation. OBJECTIVES: To evaluate the relationship between the presence/absence and widths of midsagittal tissue bridges and walking ability among veterans with cervical, predominantly chronic SCI. SETTING: University research and hospital setting. METHODS: T2-weighted midsagittal MRIs of 22 United States veterans with cervical spinal cord injuries were examined. The presence/absence of midsagittal tissue bridges were determined, and the widths of present ventral and dorsal tissue bridges were measured. Midsagittal tissue bridge characteristics were related to each participant's ability to walk based off examination of clinical documentation. RESULTS: Fourteen of the analyzed participant images revealed the presence of midsagittal tissue bridges. Ten of those individuals (71%) possessed overground walking ability. The 8 individuals with no apparent tissue bridges were all unable to walk. There was a significant correlation between walking and widths of ventral midsagittal tissue bridges (r = 0.69, 95%CI: 0.52, 0.92, p < 0.001), as well as dorsal midsagittal tissue bridges (r = 0.44, 95%CI: 0.15, 0.73, p = 0.039). CONCLUSION: The evaluation of midsagittal tissue bridges may be useful in various rehabilitation settings to help inform patients' plan of care, allocation of neuromodulatory resources, and appropriate stratification into research cohorts.

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

Combining Pseudopotential and All Electron Density Functional Theory for the Efficient Calculation of Core Spectra Using a Multiresolution Approach.

Broadly speaking, the calculation of core spectra such as electron energy loss spectra (EELS) at the level of density functional theory (DFT) usually relies on one of two approaches: conceptually more complex but computationally efficient projector augmented wave based approaches or more straightforward but computationally more intensive all electron (AE) based approaches. In this work we present an alternative method, which aims to find a middle ground between the two. Specifically, we have implemented an approach in the multiwavelet madness molecular DFT code that permits a combination of atoms treated at the AE and pseudopotential (PSP) level. Atoms for which one wishes to calculate the core edges are thus treated at an AE level, while the remainder can be treated at the PSP level. This is made possible thanks to the multiresolution approach of madness, which permits accurate and efficient calculations at both the AE and PSP level. Through examples of a small molecule and a carbon nanotube, we demonstrate the potential applications of our approach.

N-terminal phosphorylation sites of herpes simplex virus 1 ICP0 differentially regulate its activities and enhance viral replication.

The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication.

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation.

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.

NLRC4 inflammasome activation is NLRP3- and phosphorylation-independent during infection and does not protect from melanoma.

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates caspase-1 and initiates potent immune responses. Structural, biochemical, and genetic data demonstrate that NAIP proteins are receptors for bacterial ligands, while NLRC4 is a downstream adaptor that multimerizes with NAIPs to form an inflammasome. NLRC4 has also been proposed to suppress tumor growth, though the underlying mechanism is unknown. Further, NLRC4 is phosphorylated on serine 533, which was suggested to be critical for its function. In the absence of S533 phosphorylation, it was proposed that another inflammasome protein, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line and mice with S533D phosphomimetic or S533A nonphosphorylatable NLRC4. Using these models in vivo and in vitro, we fail to observe a requirement for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results clarify our understanding of the mechanism and biological functions of NAIP/NLRC4 activation.

Diurnal variation of spontaneous uterine activity in nonpregnant primates (Macaca mulatta).

Diurnal variations in the average intrauterine pressure and in the average frequency of contraction were demonstrated in four nonpregnant rhesus monkeys. Uterine activity is lowest between 0400 and 0700 hours and highest between 1400 and 1700 hours. There is statistically significant correlation between uterine activity and diurnal variation in core temperature.

Tumor-derived CSF-1 induces the NKG2D ligand RAE-1δ on tumor-infiltrating macrophages.

NKG2D is an important immunoreceptor expressed on the surface of NK cells and some T cells. NKG2D recognizes a set of ligands typically expressed on infected or transformed cells, but recent studies have also documented NKG2D ligands on subsets of host non-tumor cells in tumor-bearing animals and humans. Here we show that in transplanted tumors and genetically engineered mouse cancer models, tumor-associated macrophages are induced to express the NKG2D ligand RAE-1δ. We find that a soluble factor produced by tumor cells is responsible for macrophage RAE-1δ induction, and we identify tumor-derived colony-stimulating factor-1 (CSF-1) as necessary and sufficient for macrophage RAE-1δ induction in vitro and in vivo. Furthermore, we show that induction of RAE-1δ on macrophages by CSF-1 requires PI3K p110α kinase signaling. Thus, production of CSF-1 by tumor cells leading to activation of PI3K p110α represents a novel cellular and molecular pathway mediating NKG2D ligand expression on tumor-associated macrophages.

Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Endothelial cells express NKG2D ligands and desensitize antitumor NK responses.

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.

Aging and the role of working memory resources in visuospatial attention.

Visuospatial attention has been shown to be robust to the effects of increasing age. Nonetheless, models linking individual differences in working memory capacity to attentional performance suggest that older adults may experience disruptions in visuospatial attention under conditions of resource load. Two experiments were conducted to investigate the effects of age and concurrent working memory load on two tasks that have been proposed to require posterior attentional processes. The findings suggest that loading working memory resources selectively disrupts performance on a nonintegrated Stroop task, whereas cue utilization remains intact. In addition, imposing a working memory load delays the deployment of visuospatial attention in both experiments. Regarding the effects of age, findings suggest that older adults can effectively perform both attentional tasks despite working memory load. Age differences did emerge in the time course of cue utilization. Findings point to the resilience of visuospatial attention in aging, even under conditions of significant cognitive load. We discuss these results and their implications for models postulating a role for working memory capacity in attentional behaviors.

Immunosurveillance and immunotherapy of tumors by innate immune cells.

Increasing evidence supports a role for innate immune effector cells in tumor surveillance. Natural killer (NK) cells and myeloid cells represent the two main subsets of innate immune cells possessing efficient but quite different tumor suppressive abilities. Here, we describe the germline-encoded NK cell receptors that play a role in suppressing tumor development and describe briefly the cellular pathways leading to the upregulation of their ligands in tumor cells. We also describe mechanisms underlying the elimination of tumor cells by macrophages and a recently characterized mechanism dedicated to sensing cytosolic DNA that is implicated in antitumor immune responses.

Withdrawal from methadone maintenance. Rate of withdrawal and expectation.

One hundred twenty-seven successfully methadone-hydrochloride-maintained patients were randomly assigned to one of the following four groups and studied for 30 weeks: (1) known maintenance-patients were maintained on methadone under open conditions; (2) blind maintenance-patients were maintained on methadone under double blind conditions; (3) rapid withdrawal-patients were withdrawn under double-blind conditions at a rate of 10% of initial dose per week; (4) gradual withdrawal-patients were withdrawn under double-blind conditions at a rate of 3% of initial dose per week. Differences in dropout rates, illicit drug use, symptoms scores, and requests for study interruption indicate that withdrawal from methadone maintenance should be carried out at approximately 3% of initial dose per week. Better patient preparation also is indicated to reduce the effects of expectation.

Clinical pharmacology of phencyclidine toxicity.

Phencyclidine appears to be unique in action compared with other psychedelic drugs, and its effects are less dependent upon the individual's personality than are the effects of LSD or mescaline. The authors discuss the sensory, psychological, and behavioral symptoms of phencyclidine intoxication. Most cases are of short duration and the only treatment necessary may be observation together with minimal stimulation and diazepam. However, prolonged and severe behavioral disturbances, exaggeration of preexisting thought disorder, and serious medical complications commonly occur and must be considered in the treatment plan.

Essential fatty acid sufficiency does not preclude fat-soluble-vitamin deficiency in short-bowel syndrome.

Patients with extensive small-bowel resection may experience malabsorption and nutrient deficiencies. We evaluated the ability to absorb fat and fat-soluble vitamins in a short-gut patient. For 18 wk after stopping intravenous lipid, while consuming a low-lactose, low-fat diet, he exhibited no clinical manifestations of essential fatty acid deficiency (EFAD). Serum 20:4n-6 (20:4 omega-6) and 18:2n-6 fatty acid concentrations were normal, whereas the concentration of 20:3n-9 remained less than or equal to 0.1% of total serum fatty acids. Although serum vitamin A was normal, beta-carotene was undetectable despite oral supplementation. Prothrombin time was elevated until parenteral vitamin K was given. This patient has fat absorption adequate to prevent EFAD but inadequate absorption of fat-soluble vitamins. In patients with short bowel, the requirements for parenteral lipids and fat-soluble vitamins should be determined independently.

Folic acid, mental function, and dietary habits.

The combination of biochemical theory, case studies, and patient population surveys suggests that the water soluble vitamin, folic acid, is relevant to mental functions. Several studies demonstrate a clinical correlation between folate deficiency and psychiatric hospitalization. The role of dietary deficiency to these observations has needed further examination. After reviewing the serum folate values of 269 psychiatric hospital admissions, the incidence of serum folates below 5.9 ng./ml. was found to be greater than a control group. A dietary rating for all admissions revealed a high frequency of deficiency in all patients with no greater prevalence among the low folate group. These observations agree that psychiatric patients are more likely to have low serum folate values, and suggest that poor diet does not explain the increased likelihood.

Serum leukotriene B4 levels in patients with obstructive pulmonary disease.

Leukotriene B4 has been found to be increased in the serum of cigarette smokers and some patients with bronchial asthma, as well as in the sputum of patients with cystic fibrosis and COPD. Corticosteroids supposedly may block the formation of LTB4. To determine if the effect of CS on airway disease is by reduction in LTB4, we studied serum LTB4 levels in clinically stable patients with asthma or COPD who were treated with or without CS. The LTB4 was extracted from serum and assayed by radioimmunoassay. Serum LTB4 concentrations, expressed as the mean +/- SD, were 0.36 +/- 0.15 ng/ml in ten normal controls, 0.56 +/- 0.18 ng/ml in nine asthmatic subjects, 0.67 +/- 0.2 ng/ml in eight asthmatic subjects receiving CS, 0.81 +/- 0.19 ng/ml in seven patients with COPD, and 0.97 +/- 0.29 ng/ml in eight patients with COPD receiving CS. Serum LTB4 levels in normal controls differed significantly from all groups with COPD or asthma (p less than 0.01). Levels of LTB4 in asthmatic subjects differed from levels in patients with COPD (p less than 0.03), and levels in asthmatic subjects receiving CS differed from subjects with COPD receiving CS (p less than 0.03). Concentrations of LTB4 within either the COPD or the asthmatic groups were not lower in the patients treated with CS. We conclude that serum LTB4 concentrations are higher in COPD than in asthma or normal controls and that administration of CS is not associated with low LTB4 levels. The beneficial effects of CS in obstructive airway disease appear to be mediated by mechanisms other than reduction of LTB4.

Ambulatory blood pressure monitoring in hypertensive adolescents.

The purpose of this study was to determine the ability of ambulatory blood pressure monitoring to identify youths with chronic blood pressure elevation. Nineteen adolescent boys were studied, ten had 5-year average systolic or diastolic pressures above the 95th percentile, nine had normal pressure. A Del Mar Avionics Pressurometer III system recorded an average of 121 readings on each subject. The coefficients of variation for pressure were similar for hypertensive and normotensive individuals. During classes, eight of the ten hypertensive youths had elevated pressures in over half of the measurements. Also during these classes eight of ten hypertensive boys had average systolic or diastolic pressure above the 95th percentile, whereas only one of nine normotensive boys had average pressures above this level. We suggest that schooltime ambulatory pressures may be most useful in classifying the blood pressure trend in a youth.

Actinomycosis of the female genital tract.

Four cases of actinomycosis involving the uterus and adnexal structures are reported. In 2 cases the infection was transmitted from a ruptured appendix. Ascending actinomycosis involving the endometrium and resulting in adnexal abscesses was associated with the use of an IUD in 2 patients. This infection should be suspected in any patient who develops a pelvic abscess with an IUD in place. Culture and histologic examination of tissue removed with the IUD may be a means of early diagnosis. The nature of these infections became apparent only after serious complications developed. Each patient required several surgical procedures. The diagnosis remained unsuspected until repeated laboratory examinations detected the fungus. The difficulty encountered identifying Actinomyces israeli indicates the infection is often undetected. Gallium scans were helpful in localizing occult abscesses in 2 patients.

PIP: 4 cases of actinomycosis, treated at the University of Virginia Hospital, involved the uterus and adnexal structures. 2 cases were the result of a transferred infection from a ruptured appendix. Ascending actinomycosis involved the endometrium and resulted in adnexal abcesses for 2 patients in which infection was associated with the use of an IUD. A means of early diagnosis might be through examination of tissue removed with an IUD. The nature of these infections becomes apparent only after serious complications develop. Patients required several surgical procedures. The diagnosis may remain undetected through repeated examination in the laboratory. This difficulty in detecting Actinomyces israeli indicates that the infection may often be undetected. Gallium scans were helpful in localizing occult abscesses i n 2 patients.

Progesterone production by an ovarian granulosa cell carcinoma.

A patient with a progesterone-producing granulosa cell carcinoma is the basis of this report. Seven years after initial surgical therapy pelvic masses were palpated. At laparotomy the recurrence of tumor was confirmed, and many nonresectable metastases were discovered on the surface of the liver and on the mesentery of the bowel. An exceedingly high plasma progesterone level of 6270 pg/ml was obtained in the postoperative period. During 12 months of single agent chemotherapy with melphalan, serial plasma progesterone assays declined to 310 pg/ml. Complete tumor regression was subsequently confirmed by laparoscopy. Evaluation of progesterone levels in patients with granulosa cell tumors is recommended to determine the incidence of this finding and to further assess its value in following response to therapy.