Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma

PURPOSE: In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys. METHODS: Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus. RESULTS: With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes. CONCLUSIONS: Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.     

Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo

PURPOSE: To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival. METHODS: The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo. RESULTS: In the two-hybrid system, Protein Phosphatase 1alpha1 (PP1alpha1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1alpha1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1alpha1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1alpha1 is reported. CONCLUSIONS: The strong two-hybrid interaction between PP1alpha1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1alpha1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.     

AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization

PURPOSE: Adeno-associated viral (AAV) vectors have been used to express several different proteins in the eye. The purpose of this study was to determine whether AAV-mediated intraocular gene transfer of pigment epithelium-derived factor (PEDF) inhibits the development of choroidal neovascularization (CNV) in a murine model. METHODS: C57BL/6 mice were given intravitreous or subretinal injections of a PEDF expression construct packaged in an AAV vector (AAV-chicken beta-actin promoter-exon 1-intron 1[CBA]-PEDF) or control vector (AAV-CBA-green fluorescent protein[GFP]). After 4 or 6 weeks, the Bruch's membrane was ruptured by laser photocoagulation at three sites in each eye. After 14 days, the area of CNV at each rupture site was measured by image analysis. Intraocular levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS: Four to six weeks after intraocular injection of AAV-CBA-PEDF, levels of PEDF in whole-eye homogenates were 6 to 70 ng. The average area of CNV at sites of the Bruch's membrane rupture showed no significant difference in eyes injected with AAV-CBA-PEDF compared with uninjected eyes. In contrast, 4 to 6 weeks after intraocular injection of 1.5 x 10(9) or 2.0 x 10(10) particles of AAV-CBA-PEDF, the area of CNV at the Bruch's membrane rupture sites had significantly decreased compared with CNV area at rupture sites in eyes injected with AAV-CBA-GFP. CONCLUSIONS: These data suggest that intraocular expression of PEDF or other antiangiogenic proteins with AAV vectors may provide a new treatment approach for ocular neovascularization.     

Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension

PURPOSE: Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated. METHODS: RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay. RESULTS: RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay. CONCLUSIONS: Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of glaucoma.     

Regulatory effects of deoxycholic acid, a component of the anti-inflammatory traditional Chinese medicine Niuhuang, on human leukocyte response to chemoattractants

Niuhuang is a commonly used Chinese traditional medicine with immunoregulatory and anti-inflammatory properties. Deoxycholic acid (DCA) is a major active constituent of Niuhuang. The reaction of human leukocytes to chemoattractants is an important part of the host immune response and also plays a crucial role in the development of inflammation. We, therefore, investigated the in vitro effects of DCA on human monocyte and neutrophil responses to classic chemoattractants [fMet-Leu-Phe (fMLP), complement fraction 5a (C5a)], CC chemokine [monocyte chemoattractant protein-1 (MCP-1/CCL2)], and/or CXC chemokines [stromal cell-derived factor-1 (SDF-1alpha/CXCL12), interleukin-8 (IL-8/CXCL8)]. The results showed that DCA significantly inhibited fMLP-induced monocyte and neutrophil chemotaxis and calcium mobilization, and also blocked the binding of [3H]fMLP and anti-formyl peptide receptor (FPR) monoclonal antibodies (mAb) to the cells. The inhibitory effects of DCA on calcium mobilization and anti-FPR-mAb binding to the receptor could be abrogated by washing DCA out of the cell suspension, suggesting that DCA blocked fMLP receptors via a steric hindrance mechanism, not via receptor internalization. DCA had no significant inhibitory effects on MCP-1-, SDF-1alpha-, or C5a-induced monocyte function, or C5a- or IL-8-induced neutrophil function. Taken together, our experimental results suggest that blockade of fMLP receptors may contribute to the anti-inflammatory effects of traditional medicine containing DCA.     

Perspectives of patients with cystic fibrosis on preventive counseling and transition to adult care

The purpose of this study was to investigate how adolescents and adults with cystic fibrosis (CF) view preventive counseling and their transition to adult-centered care within a children's hospital. Thirty-two patients >/=16 years old diagnosed with CF were recruited from a pediatric tertiary care setting. During face-to-face interviews, patients were asked 27 structured questions and completed a 30-item self-administered questionnaire on preventive counseling by healthcare providers and on transition issues. The median age of patients was 25.5 years (range, 16-43 years); 69% of patients identified a pulmonologist as their "main doctor," even though 78% had a primary care provider. Participants felt that 13-16 years of age was the best time for them to begin spending time alone with their main doctor. Less than half of the participants recalled receiving preventive counseling during the previous 12 months, and more patients wanted to discuss issues than actually did. Qualitative data emphasized the importance of independence in making decisions in healthcare and establishing relationships with providers, and many patients did not desire to transfer care to an adult hospital. Participants identified adult-focused services such as inpatient rooms, discussion groups, work options, and social service support that would enhance care. In conclusion, the majority of adolescent/young adult patients with CF receiving care in a pediatric institution reported satisfaction with their healthcare. However, patients identified preventive issues that they desired to be more regularly addressed, starting in early adolescence, and changes in the delivery of services to enhance transition to adult-oriented care. This study underscored the understanding of the integration of transition planning into the facilitation of healthcare decision-making by the adolescent in issues of self-care, sexuality, education, and finances. Future initiatives to enhance the care of patients with CF should provide training of pulmonologists in preventive care and increased attention to helping patients utilize appropriate primary-care services during the adult years. In addition, prospective studies are needed to compare outcomes of CF patients who have transitioned and transferred to adult hospitals and those transitioning to adult-oriented services in a pediatric institution.     

Cellular pharmacology studies of shikonin derivatives

The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon Sieb. et Zucc.(Boraginaceae) and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Historically, Zicao root extracts have been used to treat macular eruption, measles, sore-throat, carbuncles and burns. Multiple pharmacological actions have been attributed to shikonin, e.g. antiinflammatory, antigonadotropic and anti-HIV-1 activity. In this review, several therapeutic applications of shikonin will be summarized including its pleiotropic, antiinflammatory and antitumour effects. Widely diverse and sometimes conflicting activities have been attributed to shikonin, e.g. wound healing, enhanced granuloma formation, suppression of local acute inflammatory reactions, inhibition of angiogenesis, inhibition of select chemokine ligands, inhibition of DNA topoisomerase activity, inhibition of platelet activation and antimicrobial activity. Comparison of the various reported mechanisms of action for shikonin lead us to hypothesize that shikonin is an effective inhibitor of protein-protein interaction with multiple targets in both the intracellular and extracellular compartments. This general inhibitory effect can account for the broad spectrum of shikonin biological and pharmacological activities.     

Implicit and explicit alcohol-related cognitions

This article presents the proceedings of a symposium at the 2001 RSA Meeting in Montreal, Canada organized by Reinout W. Wiers and Alan W. Stacy. The purpose of the symposium was to present recent applications of implicit cognitive processing theory to alcohol research. Basic cognitive research has demonstrated that implicit cognition influences memory and behavior without explicit recall or introspection. The presentations from this symposium show that implicit cognition approaches yield new insights into understanding drinking motivation. The presentations were: (1) An introduction by Alan W. Stacy; (2) Implicit cognition and alcohol use. Involvement of other variables? (Susan L. Ames); (3) Alcohol expectancies and the art of implicit priming (Jane A. Noll); (4) Parental alcoholism and the effects of alcohol on semantic priming (Michael A. Sayette); (5) Implicit arousal and explicit liking of alcohol in heavy drinkers (Reinout W. Wiers); and (6) Negative affective cues and associative cognition in problem drinkers (Martin Zack). Comments were provided by the discussant Marvin Krank. The presented studies demonstrated that: (1) implicit memories of alcohol associations are powerful predictors and cross‐sectional correlates of alcohol use; (2) implicit retrieval processes influence alcohol outcome expectancies and alcohol consumption; (3) alcohol consumption influences implicit memory processing; (4) heavy drinkers reveal different affective responses in implicit and explicit tasks; and (5) negative affect exerts an implicit priming effect for alcohol associations in problem drinkers. These findings illustrate the importance of implicit cognition in understanding alcohol abuse and demonstrate the potential of the theoretical framework for more widespread application across a variety of areas of alcohol research, including diagnostics for the risk of alcohol abuse, treatment, and prevention.     

Epidemiology of methicillin-resistant Staphylococcus aureus colonization in a surgical intensive care unit.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of healthcare-associated infections among surgical intensive care unit (ICU) patients, though transmission dynamics are unclear. OBJECTIVE: To determine the prevalence of MRSA nasal colonization at ICU admission, to identify associated independent risk factors, to determine the value of these factors in active surveillance, and to determine the incidence of and risk factors associated with MRSA acquisition. DESIGN: Prospective cohort study. SETTING: Surgical ICU at a teaching hospital. PATIENTS: All patients admitted to the surgical ICU. RESULTS: Active surveillance for MRSA by nasal culture was performed at ICU admission during a 15-month period. Patients who stayed in the ICU for more than 48 hours had nasal cultures performed weekly and at discharge from the ICU, and clinical data were collected prospectively. Of 1,469 patients, 122 (8%) were colonized with MRSA at admission; 75 (61%) were identified by surveillance alone. Among 775 patients who stayed in the ICU for more than 48 hours, risk factors for MRSA colonization at admission included the following: hospital admission in the past year (1-2 admissions: adjusted odds ratio [aOR], 2.60 [95% confidence interval {CI}, 1.47-4.60]; more than 2 admissions: aOR, 3.56 [95% CI, 1.72-7.40]), a hospital stay of 5 days or more prior to ICU admission (aOR, 2.54 [95% CI, 1.49-4.32]), chronic obstructive pulmonary disease (aOR, 2.16 [95% CI, 1.17-3.96]), diabetes mellitus (aOR, 1.87 [95% CI, 1.10-3.19]), and isolation of MRSA in the past 6 months (aOR, 8.18 [95% CI, 3.38-19.79]). Sixty-nine (10%) of 670 initially MRSA-negative patients acquired MRSA in the ICU (corresponding to 10.7 cases per 1,000 ICU-days at risk). Risk factors for MRSA acquisition included tracheostomy in the ICU (aOR, 2.18 [95% CI, 1.13-4.20]); decubitus ulcer (aOR, 1.72 [95% CI, 0.97-3.06]), and receipt of enteral nutrition via nasoenteric tube (aOR, 3.73 [95% CI, 1.86-7.51]), percutaneous tube (aOR, 2.35 [95% CI, 0.74-7.49]), or both (aOR, 3.33 [95% CI, 1.13-9.77]). CONCLUSIONS: Active surveillance detected a sizable proportion of MRSA-colonized patients not identified by clinical culture. MRSA colonization on admission was associated with recent healthcare contact and underlying disease. Acquisition was associated with potentially modifiable processes of care.