IL-17C amplifies epithelial inflammation in human psoriasis and atopic eczema.

BACKGROUND: Key pathogenic events of psoriasis and atopic eczema (AE) are misguided immune reactions of the skin. IL-17C is an epithelial-derived cytokine, whose impact on skin inflammation is unclear. OBJECTIVE: We sought to characterize the role of IL-17C in human ISD. METHODS: IL-17C gene and protein expression was assessed by immunohistochemistry and transcriptome analysis. Primary human keratinocytes were stimulated and expression of cytokines chemokines was determined by qRT-PCR and luminex assay. Neutrophil migration towards supernatant of stimulated keratinocytes was assessed. IL-17C was depleted using a new IL-17C-specific antibody (MOR106) in murine models of psoriasis (IL-23 injection model) and AE (MC903 model) as well as in human skin biopsies of psoriasis and AE. Effects on cell influx (mouse models) and gene expression (human explant cultures) were determined. RESULTS: Expression of IL-17C mRNA and protein was elevated in various ISD. We demonstrate that IL-17C potentiates the expression of innate cytokines, antimicrobial peptides (IL-36G, S100A7 and HBD2) and chemokines (CXCL8, CXCL10, CCL5 and VEGF) and the autocrine induction of IL-17C in keratinocytes. Cell-free supernatant of keratinocytes stimulated with IL-17C was strongly chemotactic for neutrophils, thus demonstrating a critical role for IL-17C in immune cell recruitment. IL-17C depletion significantly reduced cell numbers of T cells, neutrophils and eosinophils in murine models of psoriasis and AE and led to a significant downregulation of inflammatory mediators in human skin biopsies of psoriasis and AE ex vivo. CONCLUSION: IL-17C amplifies epithelial inflammation in Th2 and Th17 dominated skin inflammation and represents a promising target for the treatment of ISD.

Influence of the phase effect on gradient-based and statistics-based focus measures in bright field microscopy.

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient-based and statistics-based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient-based focus measures tend to yield typical focus curves, whereas statistics-based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch-wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty-four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on gamma-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

Midbrain pathways for prepulse inhibition and startle activation in rat.

The midbrain is essential for prepulse inhibition (PPI) of the startle reflex, but the exact neural circuits for PPI are not yet determined. Electrical stimulation of the superior colliculus (SC) or pedunculopontine tegmentum was used to characterize the neurons and pathways that mediate PPI and the activation of startle that also occurs at higher currents in the same sites. Startle was inhibited by prepulses in most, but not all SC sites, with the lowest intensity sites in intermediate layers of SC. PPI latencies in SC sites were 4-6 ms longer than in inferior colliculus, intercollicular nucleus or pedunculopontine sites. Contrary to previous serial models, there must be two parallel midbrain pathways for PPI, a faster auditory pathway from inferior colliculus to pedunculopontine tegmentum, and a slower multimodal SC output for PPI. Double-pulse stimulation of SC sites shows that PPI results from direct stimulation of neurons with moderate refractory periods (0.4-1.0 ms), similar to SC neurons that mediate contraversive turning responses. By contrast, startle activation occurring at higher currents in all SC sites (even sites where PPI could not be elicited) results from stimulation of very short refractory period neurons (0.3-0.5 ms) and very long refractory period neurons (1.0-2.0 ms), with startle inhibition often found from 0.5-1.0 ms. Startle activation appears to result from stimulation of short refractory period neurons in deep SC layers that mediate fear-potentiated startle, plus long refractory period substrates in more dorsal SC sites.

The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene.

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF (A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE. Here, we show that the promoter regions of the hapB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box alpha and box beta). Electrophoretic mobility shift assays (EMSAs) using both nuclear extracts and the purified, reconstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady-state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants, suggesting a repressing effect of AnCF on hapB expression. Consistently, in a hapB deletion background the hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using an inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box beta led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. These results indicate that (i) box beta is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.

Supplemental morphine infusion into the posterior ventral tegmentum extends the satiating effects of self-administered intravenous heroin.

Rats learn to self-administer intravenous heroin; well-trained animals lever-press at a slow and regular pace over a wide range of intravenous doses. The pauses between successive earned infusions are proportional to the dose of the previous injection and are thought to reflect periods of drug satiety. Rats will also self-administer opiates by microinjection directly into sites in the posterior regions of the ventral tegmentum. To determine if the pauses between self-administered intravenous injections are due to opiate actions in posterior ventral tegmentum, we delivered supplemental morphine directly into this region during intravenous self-administration sessions in well-trained rats. Reverse dialysis of morphine into the posterior ventral tegmentum increased the intervals between earned injections. The inter-response intervals were greatest for infusion into the most posterior ventral tegmental sites, sites in a region variously known as the tail of the ventral tegmental area or as the rostromedial tegmental nucleus. These sites at which morphine prolongs inter-response intervals, correspond to the sites at which opiates have been found most effective in reinforcing instrumental behavior.

Contributions of the vestibular nucleus and vestibulospinal tract to the startle reflex.

The startle reflex is elicited by strong and sudden acoustic, vestibular or trigeminal stimuli. The caudal pontine reticular nucleus, which mediates acoustic startle via the reticulospinal tract, receives further anatomical connections from vestibular and trigeminal nuclei, and can be activated by vestibular and tactile stimuli, suggesting that this pontine reticular structure could mediate vestibular and trigeminal startle. The vestibular nucleus, however, also projects to the spinal cord directly via the vestibulospinal tracts, and therefore may mediate vestibular startle via additional faster routes without a synaptic relay in the hindbrain. In the present study, the timing properties of the vestibular efferent pathways mediating startle-like responses were examined in rats using electrical stimulation techniques. Transient single- or twin-pulse electrical stimulation of the vestibular nucleus evoked bilateral, startle-like responses with short refractory periods. In chloral hydrate-anesthetized rats, hindlimb electromyogram latencies recorded from the anterior biceps femoris muscle were shorter than those for stimulation of the trigeminal nucleus, and similar to those for stimulation of the caudal pontine reticular nucleus or ventromedial medulla. In awake rats, combining vestibular nucleus stimulation with either acoustic stimulation or trigeminal nucleus stimulation enhanced the whole-body startle-like responses and led to strong cross-modal summation without collision effects. In both chloral hydrate-anesthetized and awake rats, combining vestibular nucleus stimulation with ventromedial medulla stimulation produced a symmetrical collision effect, i.e. a loss of summation at the same positive and negative stimulus intervals, indicating a continuous connection between the vestibular nucleus and ventromedial medulla in mediating vestibular startle. By contrast, combining trigeminal nucleus stimulation with ventromedial medulla stimulation resulted in an asymmetric collision effect when the trigeminal nucleus stimulation preceded ventromedial medulla stimulation by 0.5 ms, suggesting that a monosynaptic connection between the trigeminal nucleus and ventromedial medulla mediates trigeminal startle. We propose that the vestibulospinal tracts participate strongly in mediating startle produced by activation of the vestibular nucleus. The convergence of the vestibulospinal tracts with the reticulospinal tract within the spinal cord therefore provides the neural basis of cross-modal summation of startling stimuli.

Kynurenate in the pontine reticular formation inhibits acoustic and trigeminal nucleus-evoked startle, but not vestibular nucleus-evoked startle.

The startle reflex is elicited by acoustic, trigeminal or vestibular stimulation, or by combinations of these stimuli. Acoustic startle is mediated largely by ibotenate-sensitive neurons in the ventrocaudal pontine reticular formation (PnC). In these studies we tested whether startle elicited by stimulation of different modalities is affected by infusion of the non-selective glutamate antagonist, kynurenate, into the PnC. In awake rats, startle responses evoked by either acoustic or spinal trigeminal nucleus stimulation were inhibited by kynurenate, but not saline, infusions, with the most effective placements nearest PnC. In chloral hydrate-anesthetized rats, kynurenate in the PnC reduced trigeminal nucleus-evoked hindlimb EMG responses, but not vestibular nucleus-evoked startle. Kynurenate in the vestibular nucleus had no effect on trigeminal nucleus-evoked startle. These results indicate that trigeminal nucleus stimulation evokes startle largely through glutamate receptors in the PnC, similarly to acoustic startle, but vestibular nucleus-evoked startle is mediated through other pathways, such as the vestibulospinal tract.

Effects of intravitreal corticosteroids in the treatment of Bacillus cereus endophthalmitis.

OBJECTIVE: To investigate whether intravitreal corticosteroid therapy reduces the extent of inflammatory intraocular tissue damage caused by Bacillus cereus endophthalmitis. METHODS: New Zealand white rabbits were inoculated with 1 x 10(6) B cereus organisms and randomized to receive no treatment (control eyes; n=14), intravitreal vancomycin hydrochloride (n=13), or a combination of intravitreal vancomycin and dexamethasone sodium phosphate (n=13) after 24 hours. The eyes were examined and graded for clinical signs of infection and inflammation on days 7 and 14, followed by enucleation for histopathologic analysis. RESULTS: Both treated groups had significantly less clinical sequelae than controls on day 7. By day 14, eyes given combination treatment had significantly less clinically graded corneal (P=.03) and conjunctival (P=.007) inflammation than eyes treated with vancomycin. Histopathologic analysis revealed a significant decrease in inflammatory changes between all treated eyes and controls at day 14. The only statistically significant difference between eyes given combination treatment and eyes given vancomycin alone was in the retina (P=.03). CONCLUSIONS: Intravitreal corticosteroids may enhance the recovery from B cereus endophthalmitis when given in conjunction with intravitreal antibiotics. The beneficial effect of corticosteroids is noted clinically, but not histologically, by day 14 after single-dose treatment in rabbits. CLINICAL RELEVANCE: This study provides evidence that the use of intravitreal corticosteroids with antibiotics for the treatment of B cereus endophthalmitis may lead to an improvement compared with the use of antibiotics alone. Arch Ophthalmol. 2000;118:803-806

Selective alpha 2-adrenergic agonists B-HT 920 and UK14304-18. Effects on aqueous humor dynamics in monkeys.

Selective alpha 2-adrenergic agonists UK14304-18 and B-HT 920 were evaluated in the eyes of cynomolgus monkeys. In normal monkeys, unilateral topical application of 0.3%, 0.5%, or 1% UK14304-18 or B-HT 920 reduced (P less than .05) intraocular pressure bilaterally up to 9.9 +/- 1.2 mm Hg (mean +/- SEM) and 8.4 +/- 1.4 mm Hg in treated and contralateral eyes, respectively. Five-day twice-daily 0.5% UK14304-18 administration reduced (P less than .05) intraocular pressure up to 49% in eight glaucomatous monkeys. In eight normal monkeys, 0.5% B-HT 920 and 0.5% UK14304-18 produced no alterations in outflow facility. Following unilateral application of 0.5% B-HT 920 or 0.5% UK14304-18, fluorophotometrically measured aqueous humor production was reduced (P less than .05) bilaterally up to 67% compared with baseline values. Also, 0.5% UK14304-18 reduced (P less than .025) systolic and diastolic blood pressure. UK14304-18 and B-HT 920 seem to reduce intraocular pressure by decreasing aqueous production. They are potential new agents for the treatment of glaucoma.

Conditioned brain-stimulation reward attenuates the acoustic startle reflex in rats.

The acoustic startle reflex (ASR) in rats is attenuated by a light paired with food or, in humans, by "pleasant" pictures. Rats were trained to barpress for lateral hypothalamus (LH) stimulation. ASR amplitudes were then measured at 4 intensities, with or without a light. Control rats that did not receive brain-stimulation reward (BSR) showed initially lower ASR amplitudes than did rats exposed to BSR, but both groups responded similarly with or without light. Next, experimental rats were given BSR in the presence of light but not in its absence. After conditioning, ASR amplitudes were reduced, and ASR thresholds were raised by a mean of 2.6 dB in the light but remained at preconditioning levels without light. No such change was found for control rats or rats with placements outside the LH.

Macular complications associated with posterior staphyloma.

PURPOSE: To report macular abnormalities associated with posterior staphyloma in eyes with myopia. METHODS: In a retrospective study, we surveyed 116 eyes of 58 patients with myopic refractions. Myopic fundus abnormalities are related to clinically quantified posterior staphyloma formation. RESULTS: A posterior staphyloma was present in 88 (75.9%) of 116 eyes with myopic refractions of -3 diopters or more. Best-corrected visual acuity was decreased among eyes in all staphyloma grades. Eyes with the shallowest staphyloma depth (grade 1) displayed the largest drop in visual acuity as well as the greatest frequency of choroidal neovascular membranes and hemorrhages. A linear relationship was observed between staphyloma grade and conus formation (P = .001), retinal pigment epithelial defects (P = .0001), lacquer cracks (P = .0001), and chorioretinal atrophy (P = .001). All these variables were increased in staphylomatous eyes. A significant difference in means by staphyloma grade was observed for myopic refractive error (P = .001), axial length (P = .001), and best-corrected visual acuity (logMAR, P = .0001). CONCLUSIONS: There was an unexpected high frequency of choroidal neovascular membranes, hemorrhage, and poor best-corrected visual acuity in the lower staphyloma categories. This suggests that the development of a choroidal neovascular membrane requires relative preservation of the choriocapillaris as present in eyes with less advanced stages of posterior staphyloma formation.

Survival differences associated with treatment of cytomegalovirus retinitis in Maryland patients with AIDS, 1987-1994.

Differences in survival related to treatment of cytomegalovirus (CMV) retinitis in AIDS patients were studied. The medical records of adult AIDS patients who had been diagnosed with CMV retinitis in a Maryland inpatient facility between September 1987 and September 1994 were reviewed to assess determinants of survival, including treatment with ganciclovir and foscarnet, use of zidovudine, and demographic characteristics. The review was based on inpatient and outpatient medical records and computerized data from the Maryland HIV Information System. Of 212 AIDS patients with CMV retinitis, 123 (58.0%) were treated exclusively with ganciclovir, 55 (25.9%) received foscarnet only, and the remaining 34 (16.1%) received both ganciclovir and foscarnet at some point after their diagnosis for CMV retinitis. Patients who received both drugs survived significantly longer after the diagnosis than patients who received either drug by itself. The median time from diagnosis of CMV retinitis to death was 464 days for patients receiving both drugs, 225 days for ganciclovir recipients, and 202 days for foscarnet recipients. Other positive predictors of survival were male sex and use of zidovudine. Among Maryland adults with AIDS who were treated for CMV retinitis between September 1987 and September 1994, the most common treatment for the eye infection was ganciclovir. Patients receiving both ganciclovir and foscarnet survived longer than those treated with either drug alone.

Efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in age-related macular degeneration.

PURPOSE: To determine the efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in patients with age-related macular degeneration (ARMD). METHODS: We conducted a retrospective review of patients with ARMD treated with TTT from June, 1999 through July, 2000 at a retina referral practice. TTT was delivered through a slit-lamp using a modified diode laser at 810 nm wavelength and a spot size of 3 mm delivered at one location for a minimum of 60 seconds duration. Re-treatment was performed at 2-month intervals if indicated. RESULTS: 81 eyes of 77 patients were included in the study. Vision improved greater than one line Snellen in 18 eyes (22%), vision was stable within one line Snellen in 38 (47%), and worsened greater than one line Snellen in 25 (31%). Patients had a mean follow-up of 9 months. The average number of treatments was 1.37 (range 1 to 4). Pretreatment vision was less than or equal to 20/200 in 54% of eyes. CONCLUSIONS: Transpupillary thermotherapy may stabilize visual acuity in a majority of patients with occult subfoveal choroidal neovascularization secondary to ARMD. Proof of therapeutic benefit is best determined by a randomized clinical trial that is currently underway (TTT4CNV).

Race and the treatment of cytomegalovirus retinitis in a cohort of patients with acquired immunodeficiency syndrome.

This historical cohort study assessed the impact of race on critical factors in the diagnosis and drug treatment of cytomegalovirus (CMV) retinitis in acquired immunodeficiency syndrome (AIDS) patients over a 7-year period. The study subjects included 194 adult patients with a history of AIDS who were treated for CMV retinitis between September 1987 and September 1994. Abstracted inpatient hospital medical records and a statewide automated AIDS database were the primary sources of data. Patients were assessed for severity of CMV retinitis at diagnosis, time from initial CMV retinitis diagnosis to first treatment, survival from diagnosis of AIDS, and initiation of drug treatment for CMV retinitis. Results indicated a significant difference in the severity of CMV retinitis at diagnosis by race. Patients diagnosed with early disease were more likely to be white, whereas patients diagnosed with severe disease were more likely to be black. There was no difference in the type of CMV retinitis treatment or patient survival time after diagnosis, nor time to treatment once diagnosed by race. These results suggest that differences in survival may not be the result of discrimination against black patients and may be due more likely to practices associated with accessing medical treatment.

Penetrating eye injury in war.

The percentage of penetrating eye injuries in war has increased significantly in this century compared with the total number of combat injuries. With the increasing use of fragmentation weapons and possibly laser weapons on the battle-field in the future, the rate of eye injuries may exceed the 13% of the total military injuries found in Operations Desert Storm/Shield. During the Iran-Iraq War (1980-1988), eye injuries revealed that retained foreign bodies and posterior segment injuries have an improved prognosis in future military ophthalmic surgery as a result of modern diagnostic and treatment modalities. Compared with the increasing penetrating eye injuries on the battlefield, advances in ophthalmic surgery are insignificant. Eye armor, such as visors that flip up and down and protect the eyes from laser injury, needs to be developed. Similar eye protection is being developed in civilian sportswear. Penetrating eye injury in the civilian sector is becoming much closer to the military model and is now comparable for several reasons.