A Low Concentration of Tacrolimus/Semifluorinated Alkane (SFA) Eyedrop Suppresses Intraocular Inflammation in Experimental Models of Uveitis.

PURPOSE: Corticosteroids remain the mainstay therapy for uveitis, a major cause of blindness in the working age population. However, a substantial number of patients cannot benefit from the therapy due to steroids resistance or intolerance. Tacrolimus has been used to treat refractory uveitis through systemic administration. The aim of this study was to evaluate the therapeutic potential of 0.03% tacrolimus eyedrop in mouse models of uveitis. METHODS: 0.03% tacrolimus in perfluorobutylpentane (F4H5) (0.03% Tacrolimus/SFA) was formulated using a previously published protocol. Tacrolimus suspended in PBS (0.03% Tacrolimus/PBS) was used as a control. In addition, 0.1% dexamethasone (0.1% DXM) was used as a standard therapy control. Endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) were induced in adult C57BL/6 mice using protocols described previously. Mice were treated with eyedrops three times/day immediately after EIU induction for 48 h or from day 14 to day 25 post-immunization (for EAU). Clinical and histological examinations were conducted at the end of the experiment. Pharmacokinetics study was conducted in mice with and without EIU. At different times after eyedrop treatment, ocular tissues were collected for tacrolimus measurement. RESULTS: The 0.03% Tacrolimus/SFA eyedrop treatment reduced the clinical scores and histological scores of intraocular inflammation in both EIU and EAU to the levels similar to 0.1% DXM eyedrop treatment. The 0.03% Tacrolimus/PBS did not show any suppressive effect in EIU and EAU. Pharmacokinetic studies showed that 15 min after topical administration of 0.03% Tacrolimus/SFA, low levels of tacrolimus were detected in the retina (48 ng/g tissue) and vitreous (2.5 ng/ml) in normal mouse eyes, and the levels were significantly higher in EIU eyes (102 ng/g tissue in the retina and 24 ng/ml in the vitreous). Tacrolimus remained detectable in intraocular tissues of EIU eyes 6 h after topical administration (68 ng/g retinal tissue, 10 ng/ml vitreous). Only background levels of tacrolimus were detected in the retina (2-8 ng/g tissue) after 0.03% Tacrolimus/PBS eyedrop administration. CONCLUSION: 0.03% Tacrolimus/SFA eyedrop can penetrate ocular barrier and reach intraocular tissue at therapeutic levels in mouse eyes, particularly under inflammatory conditions. 0.03% Tacrolimus/SFA eyedrop may have therapeutic potentials for inflammatory eye diseases including uveitis.

Role of oxidative tissue injury in the pathophysiology of experimentally induced equine laminitis: a comparison of 2 models.

BACKGROUND: Oxidative stress reportedly plays a role in sepsis-induced organ dysfunction and failure in many species. In septic horses, laminae are targeted; evidence of laminar oxidative stress has been reported experimentally in the black walnut extract (BWE) model. Carbohydrate (CHO)-induced laminitis may be more similar to clinical sepsis-related laminitis than the BWE model in that animals with CHO-induced disease commonly develop laminar failure. The role of oxidative stress in the CHO model remains unknown. HYPOTHESIS/OBJECTIVES: Markers of oxidative stress will be increased in laminae from horses with BWE- and CHO-induced laminitis. ANIMALS: Banked laminar tissue from various time points from animals subjected to BWE (n = 15) and CHO (n = 20) protocols. METHODS: Laminar 4-hydroxynonenal (4-HNE) and protein carbonyl content were evaluated by slot blot analysis. Laminar 3-nitrotyrosine (3-NT) immunohistochemistry was performed. RESULTS: The number of laminar 3-NT (+) cells was increased at developmental and Obel grade 1 (OG1) time points in the BWE model (versus control [CON]; P= .013) and lower in OG1 tissues than CON in the CHO model (P = .04). No change in 4-HNE content was observed in the CHO model, and no increase in laminar protein carbonyl content was present in either model (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: These results do not support a prominent role for oxidative stress at examined time points in CHO-overload laminitis and support transient oxidative stress in the BWE model. Tissue oxidation does not appear to be a central early pathophysiologic event in CHO-associated laminitis.

Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen-associated molecular pattern motifs.

REASONS FOR PERFORMING STUDY: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. OBJECTIVES: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen-associated molecular pattern molecules (PAMPs) from both Gram-negative and positive bacteria likely to be present in equine sepsis. METHODS: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time-quantitative PCR was used to quantify interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and CXCL8 mRNA concentrations. RESULTS: Increases (P<0.05) in IL-1beta, IL-6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL-6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. CONCLUSIONS: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram-negative sepsis and unresponsive to PAMPs most commonly associated with Gram-positive sepsis. POTENTIAL RELEVANCE: The increased incidence of injury of epidermal structures in clinical cases of Gram-negative (vs. Gram-positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram-positive PAMPs in the equine keratinocyte.

Tissue concentrations of 4-HNE in the black walnut extract model of laminitis: indication of oxidant stress in affected laminae.

In the septic horse prone to laminitis, a similar activation of the innate immune system appears to occur as reported in the septic human prone to organ failure. Because oxidant injury plays a central role in organ failure occurring due to an overzealous innate immune response in human sepsis, this study was performed to determine whether there was evidence of oxidant stress in the laminar tissue in the early stages of laminitis. 4-Hydroxy-2-nonenal (4-HNE), a lipid aldehyde that forms due to lipid peroxidation occurring during episodes of oxidant stress, readily forms adducts with cellular proteins; these adducts can be assessed as a marker of oxidant stress in the form of lipid peroxidation. In this study, a slot blot technique was used to assess 4-HNE adduct concentrations in the laminae, lung, liver, and intestinal tract in the black walnut extract (BWE) model of laminitis. Significant increases in laminar 4-HNE adduct concentrations were identified at two early stages in the BWE model, in the absence of such changes in the other tissues. These data indicate that oxidant stress may play an important role in the laminar failure in laminitis, and further support the concept that a poor antioxidant response in the laminae relative to other equine tissues may be responsible for failure of the laminae in the septic horse. In contrast, tissues such as the lung and liver that undergo oxidant injury in human sepsis appear to be relatively protected in horses.

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response.

REASONS FOR PERFORMING STUDY: Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS: Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS: Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS: Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE: These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.

Intractable tinnitus and sensorineural deafness cured by surgical correction of coarctation of aorta.

Tinnitus is a challenging condition to manage with no effective treatment. We report a case of lifelong tinnitus present in a patient who was cured with the surgical repair of his coarctation of aorta. The patient was a fit 37-year-old with coarctation of aorta, senserineural deafness and intractable tinnitus. He underwent a thoracotomy and tube by-pass of his coarctation of aorta. A complete relief of his tinnitus and improvement in his hearing was noted postoperatively and confirmed on audiograms. This is the first reported case of tinnitus due to coarctation of aorta relieved by surgical correction of the coarctation.

The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.

Alternative and complementary therapies in nursing curricula.

The growing interest in and use of alternative and complementary therapies by health care professionals and laypersons, the incorporation of these therapies in medical curricula and practice, and the greater acceptance of the legitimacy of such treatment methods require that nurse educators consider how this content may be incorporated into curricula. The authors propose that prelicensure students learn the premises that support such healing practices and develop skill in eliciting and evaluating patients' use of alternative therapies. In-depth study of one or more complementary therapies may be the focus of continuing education or elective courses. Students in graduate programs may investigate the safety and efficacy of nonorthodox therapies.

Complementary, alternative, integrative: have nurses kept pace with their clients?

While medical literature reflects an interest in the use of complementary therapies, there is a paucity of studies in the nursing literature addressing the use of therapies by nurses, either on themselves or on their clients. While the utilization rate of complementary therapies by the general population has been estimated to be as high as 45%, and nurses are interacting with clients who use these therapies on a daily basis, little is known about nurses' attitudes, knowledge, or perceived efficacy of the therapies.

Complementary, alternative, integrative: have nurses kept pace with their clients?

While medical literature reflects an interest in the use of complementary therapies, there is a paucity of studies in the nursing literature addressing the use of therapies by nurses, either on themselves or on their clients. While the utilization rate of complementary therapies by the general population has been estimated to be as high as 45%, and nurses are interacting with clients who use these therapies on a daily basis, little is known about nurses' attitudes, knowledge, or perceived efficacy of the therapies.

Alterations in dopamine clearance and catechol-O-methyltransferase activity by dopamine infusions in children.

OBJECTIVE: To determine the role of catechol-O-methyltransferase (COMT) in the biodisposition of pharmacologic concentrations of dopamine. DESIGN: The study was an open-label dose escalation trial in which dopamine was employed as the sole exogenous catecholamine. The dosage was adjusted to achieve improvements in cardiac output or to augment renal function. SETTING: A 16-bed pediatric intensive care unit serving both medical and surgical patients. PATIENTS: The study was performed using 14 dopamine-treated and five untreated control patients. Children ranged in age from 16 days to 12 yrs; five of the treated patients and two of the untreated controls were female. All but one of the study patients were enrolled within 24 hrs of palliative or corrective surgery for congenital heart disease. Control patients had noncardiac surgical procedures. Both treated and control groups were similar with respect to severity of illness, as judged by Therapeutic Intervention Scoring System score. INTERVENTIONS: All treated patients received dopamine as a continuous intravenous infusion. Infusion rates were determined by caregivers and ranged from 3.0 to 20 micrograms/kg/min. MEASUREMENTS AND MAIN RESULTS: Serial, timed blood samples were obtained from patients and control subjects for the determination of plasma dopamine concentrations and for the determination of mononuclear cell COMT activity. Measured rates of dopamine infusion (3.0 to 18.3 micrograms/kg/min) were consistently less than the nominal rates (3.0 to 20.0 micrograms/kg/min) of infusion (p < .0001) due in part to calculations based on the hydrochloride salt rather than dopamine base. At similar steady-state infusion rates, plasma dopamine concentrations varied over a four-fold range, with steady-state concentrations at even the lowest infusion rate exceeding endogenous concentrations by at least ten-fold. Variations in steady-state plasma dopamine concentration reflected large age-associated variations in dopamine clearance, which was found to be saturable at concentrations of > 200 ng/mL. Mononuclear cell COMT activity was assessed simultaneously in these patients. Baseline COMT activity varied over a six-fold range and was unrelated to dopamine clearance or patient age. COMT activity increased two- to six-fold in dopamine-treated patients with plasma steady-state dopamine concentrations of > 100 ng/mL. CONCLUSIONS: These data demonstrate marked age and concentration-dependent differences in dopamine clearance that account for large interindividual differences in the steady-state plasma dopamine concentrations in patients receiving similar infusion rates. While concomitant variability in COMT activity is observed, the lack of correlation between dopamine clearance and COMT activity suggests that COMT is not rate-limiting for the clearance of exogenously administered dopamine.

Brainstem auditory evoked response development in preterm and term baboons (Papio hamadryas).

Brainstem auditory evoked responses were recorded longitudinally from 11 neonatal baboons (Papio hamadryas), 6 of which were preterm. Recordings were made in unsedated animals from day 161 to day 362 after conception (term = 182 days). The pattern of development of both waveform morphology and of wave latency was consistent with that seen in the human neonate, with a rapid maturation of the response during the perinatal period, and then a slower development to adult values. Brainstem conduction time was measured from the wave I to wave IV interval, and this demonstrated a similar pattern, with a rapid decrease in latency up to term, and then decreasing more slowly to reach adult values by 4 months of age in the baboon.

The effect of bicarbonate-free artificial cerebrospinal fluid on spontaneous oscillations of the membrane potential in inferior olivary neurons of the rat.

The membrane potential of mature inferior olivary (IO) neurons oscillates spontaneously at frequencies up to about 10 Hz. This behavior has been attributed to the complement of membrane conductances in these cells and electronic coupling of the neurons via dendro-dendritic gap junctions. In this study intracellular recordings of transmembrane potentials were made in 52 neurons in brainstem slices from rats aged 8-23 days postnatal. During the recordings in 31 neurons the extracellular solution was exchanged from a bicarbonate- to a tris-buffered solution with constant pH. In all cells the spontaneous oscillations of the membrane potential ceased within a few minutes and in 14 of these cells the oscillations resumed with re-exposure to bicarbonate-buffered solution. The spontaneous oscillations in another 6 neurons also ceased when they were exposed to bicarbonate-buffered solution containing 10 mmol NH4Cl. These experimental manipulations produce a rise in intracellular pH despite constant extracellular pH. The low- and high-threshold potentials associated with voltage-sensitive calcium conductances in these neurons and the large hyperpolarization that follows these potentials were unaffected by substitution of the extracellular solution. However, the anomalous rectification of the membrane potential in these neurons (which could be abolished by exposure of the neurons to 2 mM CsCl) was significantly increased by 12.6% in the tris-buffered solution. The data are consistent with the hypothesis that the continuity of oscillations of the membrane potential in IO neurons depends on the transfer of current between the dendro-dendritic junctions and the soma of each neuron. This transfer is reduced when rectifying K+ conductances in the neurons are increased and the ensemble properties of the group of neurons, and the membrane potential oscillations, are lost.

A comparison between scores on Kirton's inventory for nursing students and a general student population.

This study compared scores on the Kirton Adaption-Innovation Inventory of 60 first-year nursing students with scores of 73 nonnursing majors of approximately the same age to test the hypothesis that, in general, individuals selecting nursing as a major tend to show a more adaptive style of creativity in problem solving than their nonnursing peers. Analysis indicated the nursing students were significantly more "adaptive" in problem solving and less "innovative" than the nonnursing control group.

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells.

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-L-methionine and magnesium chloride in sodium-potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-L-methionine using this method are similar to values reported previously (0.51 and 14 microM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2-3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.