Further studies on the role of IL-12 in Pseudomonas aeruginosa corneal infection.

PURPOSE: Previous studies have shown that in Pseudomonas aeruginosa ocular infection, IL-12 drives a Th1 T-cell response and IFN-gamma production in susceptible (cornea perforates) C57BL/6 (B6) mice, and that after similar infection of resistant (cornea heals) BALB/c mice, no IL-12 is detectable in cornea at either the mRNA or protein levels. Therefore, the purpose of this study was to test whether BALB/c mice are capable of responding to exogenous IL-12 administration, and whether disease responsiveness following P. aeruginosa challenge is modified. METHODS: Immunostaining, RT/PCR, recombinant cytokine injection, and histopathology were used. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. RESULTS: Injection of BALB/c mice with recombinant (r) IL-12 converted these normally resistant animals to the susceptible phenotype as evidenced by corneal perforation within 5-7 days after infection. RT-PCR analysis of the corneas of rIL-12 vs PBS/BSA-treated mice showed a significant increase in IFN-gamma and TNF-alpha mRNA levels in the rIL-12 vs PBS/BSA (vehicle)-treated mice at 3 and 5 days p.i. In addition, similar analysis of IL-4 mRNA levels showed decreased amounts of the cytokine in rIL-12 vs vehicle-treated mice. Injection of rIL-4 into susceptible B6 mice, however, failed to rescue these animals from corneal perforation following P. aeruginosa challenge. CONCLUSIONS: These data provide evidence that BALB/c mice can respond to exogenous IL-12, that the cytokine promotes susceptibility by increasing IFN-gamma and TNF-alpha production, with a concomitant reduction in IL-4 levels; and that injected rIL-4 fails to rescue susceptible B6 mice from corneal perforation after bacterial challenge.

MIP-1alpha regulates CD4+ T cell chemotaxis and indirectly enhances PMN persistence in Pseudomonas aeruginosa corneal infection.

The role of macrophage inflammatory protein-1alpha (MIP-1alpha) in cell infiltration into Pseudomonas aeruginosa-infected cornea and subsequent disease was examined. Greater amounts of the chemokine (protein and mRNA) were found in the infected cornea of susceptible B6 ("cornea perforates") versus resistant BALB/c ("cornea heals") mice from 1 to 5 days postinfection. Treatment of BALB/c mice with recombinant (r) MIP-1alpha exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea. Treatment of BALB/c mice with rMIP-1alpha also induced recruitment of activated CD4+ T cells into the affected cornea, converting resistant to susceptible mice. Depleting CD4+ T cells in r-treated BALB/c mice significantly decreased PMNs in cornea tissue, suggesting that T cells regulate persistence of PMNs at this site. In B6 mice, administration of neutralizing MIP-1alpha polyclonal antibody also significantly reduced PMN numbers and pathology. Collectively, evidence is provided that MIP-1alpha directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.

Aging and PMN response to P. aeruginosa infection.

PURPOSE: Alterations in immune system function associated with aging may contribute to increased morbidity in this population of individuals. The current studies were performed to determine aging-related changes in polymorphonuclear neutrophil (PMN) function after corneal infection with Pseudomonas aeruginosa. METHODS: Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P. aeruginosa. In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment. RESULTS: Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times. In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times. The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times. Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease. CONCLUSIONS: These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P. aeruginosa challenge. Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.

Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation.

The kinetics of IL-1 (alpha and beta) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1alpha and -1beta (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i. ). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1alpha and -1beta (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1beta was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1beta polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.

Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection.

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.

Complement defects in aged mice compromise phagocytosis of Pseudomonas aeruginosa.

PURPOSE: The role of complement in phagocytosis and killing of P. aeruginosa was examined using serum from aged vs young donor mice. METHODS: Phagocytosis, complement hemolytic and microbicidal assays were used. RESULTS: Serum from young donor mice contained a heat-labile factor which significantly enhanced phagocytic activity of cells from young mice compared with similarly treated aged donor serum. Use of cobra venom factor (CVF) to destroy C3 and the terminal complement components in serum from young or aged donor mice also significantly decreased the phagocytic activity of young cells. EGTA treatment of young or aged donor serum, to activate the alternative pathway and selectively inhibit activation of the classical pathway, resulted in a significant decrease in phagocytosis by young cells in the presence of donor serum from either group. Alternative pathway mediated hemolysis also was measured and was significantly reduced in aged vs young donor serum. PMN microbicidal activity was tested using cells from young mice in the presence of aged vs young donor serum, but no significant differences were noted. CONCLUSION: These data provide evidence that defects in the alternative pathway of complement in the serum of aged animals lead to decreased phagocytic activity of cells from young mice, but not impaired bacterial killing.

Pseudomonas aeruginosa keratitis in knockout mice deficient in intercellular adhesion molecule 1.

In this study, the role of intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of Pseudomonas aeruginosa keratitis was examined by using inbred ICAM-1-deficient knockout mice. These mice had significantly less (P

Experimental neodymium:YAG laser damage to acrylic, poly(methyl methacrylate), and silicone intraocular lens materials.

PURPOSE: To compare neodymium:YAG (Nd:YAG) laser effects on acrylic, silicone, and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) polymers. METHODS: Ten Nd:YAG laser exposures were produced in each of 6 implantation-quality acrylic (Alcon MA60BM), silicone (Staar AQ1016), and PMMA (Alcon MC60BM) IOLs under identical conditions. Each polymer type was irradiated at 6 power settings (0.3, 0.5, 1.0, 1.5, 2.0, and 3.0 mJ) and at 2 focal points (midpoint of lens optic and on the posterior surface to which a cellophane membrane was affixed). The linear extent of the damage was measured using light microscopy. Specimens exposed to 1.0 mJ were processed for scanning electron microscopy. RESULTS: The damage threshold (> or = 5 microns depth) was 0.3 mJ for silicone and 1.0 mJ for acrylic and PMMA IOLs. At the clinically relevant power levels, 1.0 to 2.0 mJ, the depth of damage in the acrylic polymer was 11.9 to 30.5 times less than the depth in the silicone polymer. Similarly, the depth of damage in the PMMA polymer was 5.4 to 52.6 times less than the depth in the silicone polymer. The morphologic pattern of damage in the silicone IOL showed a deep, irregularly configured trough with meandering tendrils. Acrylic IOL damage morphology consisted of an ameboid-shaped entry site without radiating fractures and mild posterior penetration. Poly(methyl methacrylate) IOL damage consisted of a shallow focal trough with radiating fractures. CONCLUSIONS: The silicone IOL polymer had the lowest threshold for laser-induced damage and greater linear extension of damage than the PMMA and acrylic IOL polymers. Poly(methyl methacrylate) and silicone polymers exhibited collateral damage or ejected particulates adjacent to the entry site, whereas the acrylic polymer showed a discrete locus of damage.

Viscoelastic adherence to corneal endothelium following phacoemulsification.

PURPOSE: To compare the residual adherence of viscoelastics to the corneal endothelium following phacoemulsification in an in vitro rabbit model. SETTING: Departments of Ophthalmology and Anatomy, Wayne State University, Detroit, Michigan, USA. METHODS: Three groups of 10 rabbit eyes each had a lensectomy via phacoemulsification using sodium hyaluronate (Amvisc Plus, Healon GV) or sodium chondroitin sulfate-sodium hyaluronate (Viscoat) as the viscoelastic agent. After phacoemulsification and cortex removal, a central corneal block was excised, cryofixed, and processed for light and electron microscopy. Viscoelastic thickness was determined by a calibrated reticule on the light microscope or a calibrated measuring program in the electron microscope. The nonparametric statistical test, Kruskal-Wallis, was used to compare viscoelastic groups. RESULTS: Median phacoemulsification time between viscoelastic agents was not significantly different. Median viscoelastic thicknesses were 13.0 microns for Amvisc Plus, 0.4 micron for Healon GV, and 375.0 microns for Viscoat. Each was significantly different from the others (Kruskal-Wallis, P < .001). CONCLUSIONS: Median thickness of Amvisc Plus, Healon GV, and Viscoat remaining adherent to the corneal endothelium after phacoemulsification was markedly different. Viscoat provided the greatest amount of viscoelastic material adjacent to the corneal endothelium.

Proinflammatory cytokine deficiency and pathogenesis of Pseudomonas aeruginosa keratitis in aged mice.

Corneal clarity in young adult Swiss (HSD:ICR) mice is restored after Pseudomonas aeruginosa infection. Previous data showed that this response involves a rapid up-regulation of constitutive intercellular cell adhesion molecule-1 (ICAM-1) and migration of inflammatory cells into the cornea. In contrast, in aged mice, there is no up-regulation of corneal ICAM-1, inflammatory cell infiltration into the cornea is delayed, and the cornea perforates. Therefore, the aim of this study was to test whether specific cytokines which up-regulate ICAM-1 expression differ in young and aged mice. Corneas of young (6- to 8-week-old) and aged (1- to 2-year-old) mice were scarified and inoculated with P. aeruginosa. The eyes were graded for pathologic changes (score 0 to +4); at 6, 12, 24, and 48 h postinfection (p.i.), six mice from each age group were sacrificed. Three corneas from each respective group were excised for quantitation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and gamma interferon (IFN-gamma) by enzyme-linked immunosorbent assay. The remaining three corneas from each age group were harvested for quantitation of viable bacteria by direct plate count determination and for infiltrating polymorphonuclear leukocytes (PMNs) by a myeloperoxidase (MPO) assay. Compared to those of young mice, the corneas of infected aged mice had less IL-1beta at 6 h p.i. (P < or = 0.04) and less IFN-gamma at 12 to 48 h p.i. (P < or = 0.05). Also, compared to those of young mice, corneas of aged mice had fewer PMNs (P < or = 0.008) by the MPO assay at 6 h p.i. and more viable bacteria (P < or = 0.01) per cornea by plate count determination at 24 h p.i. These data suggest that the lack of up-regulation of ocular ICAM-1 in aged mice may reflect a reduction in both IL-1beta and IFN-gamma levels in the infected cornea. Consequently, a sufficient number of PMNs and other inflammatory cells fail to rapidly migrate into the infected corneas of aged mice, the bacterial load is initially greater than that in young mice, and the cornea perforates.

Aged mice fail to upregulate ICAM-1 after Pseudomonas aeruginosa corneal infection.

PURPOSE: In young Swiss (HSD:ICR) outbred mice, corneal clarity is restored after Pseudomonas aeruginosa ocular infection, whereas disease in aged outbred mice progresses to corneal perforation. This study was conducted to elucidate further the mechanism responsible for this age-related disparity in disease response. METHODS: Corneas of young (6 to 8 weeks of age) and aged (1.5 to 2 years) female mice were scarified and inoculated with 1.0 x 10(8) colony-forming units of P. aeruginosa ATCC 19660. Eyes were scored for corneal pathology (0 to +4) at 6, 12, 24, 48, 72, 96, and 120 hours after infection. At each time point, six mice were killed from each age group, and both eyes were enucleated. Eyes (three infected, three uninfected) were embedded in OCT compound, frozen in liquid nitrogen, sectioned on a cryostat, and stained for ICAM-1 and LFA-1 immunoreactivity. The remaining six eyes (three infected, three uninfected) were embedded in eponaraldite resin, thick sectioned, and stained for light microscopic histopathologic examination. RESULTS: Immunostaining of slight to moderate intensity for ICAM-1 was seen on conjunctival fibroblasts, stromal keratocytes, corneal epithelium, and endothelium and conjunctival blood vessel endothelium of uninfected contralateral eyes in both age groups. In response to P. aeruginosa infection, only young animals were capable of upregulating ICAM-1 (as evidenced by an increase in the intensity of immunostaining) on these cells when compared to aged mice. Conversely, the intensity of immunostaining for LFA-1, a ligand for ICAM-1 on infiltrating leukocytes, was similar despite animal age. On gross observation, corneal pathology was more severe in young mice 24 to 96 hours after infection. Histopathologically, in contrast to young mice, eyes of aged animals 24 to 48 hours after infection had significantly fewer inflammatory cells, such as polymorphonuclear leukocytes (PMNs), infiltrating the corneal stroma and adhering to the endothelium near wound sites. CONCLUSION: These data suggest that the disparate response to ocular P. aeruginosa infection in young versus aged mice is due, at least in part, to the inability of aged animals to upregulate ICAM-1 above constitutively expressed levels. Consequently, the migration of inflammatory cells (PMNs) into infected corneas of aged mice is delayed, perhaps facilitating bacterial growth and contributing to a poor prognosis.

Suicidal behavior in children and adolescents with mental retardation.

Despite increasing attention to psychiatric disorders in the mentally retarded, suicidal behavior remains an underreported phenomenon in this population, particularly in children and adolescents. This study was aimed at documenting the existence of suicidal behavior among 90 consecutive admissions to a specialty unit for dually diagnosed children and adolescents in a medical school-affiliated children's psychiatric hospital. Archival chart review yielded a total of 19 patients, or 21%, for whom suicidal behavior was a presenting complaint upon admission or during hospitalization. Suicidality was distributed across gender, level of mental retardation, and psychiatric diagnosis. Additional findings of note with regard to family dysfunction and/or abuse history are summarized. Clearly, in this sample, children and adolescents with mental retardation were capable of formulating and engaging in potentially fatal acts. Results of this study suggest that suicidal behavior is an underrecognized, yet significant phenomenon in children and adolescents with mental retardation and psychiatric disorder.

An observationally based rating scale for affective symptomatology in child psychiatry.

There is growing dissatisfaction with current methods for rating affective symptoms in children. We report findings from a preliminary psychometric study of an alternative approach, that of direct observational ratings. The Emotional Disorders Rating Scale (EDRS) is an observation-based instrument containing 59 items divided into eight subscales. The results of this study indicate that measurement of nonverbal components of affective symptoms in children is feasible. Interrater reliability and internal consistency of the EDRS subscales were high. The EDRS also has potential as a measurement of state-related changes in affective behavior and as a technique for examining treatment response.

A case report of naltrexone treatment of self-injury and social withdrawal in autism.

The endogenous opiate release theory of self-injurious behavior (SIB) was investigated through double-blind placebo-controlled administration of naltrexone hydrochloride (Trexan) to a 14-year-old autistic and mentally retarded male for treatment of severe SIB. Results yielded a marked decrease in SIB during two phases of active drug treatment, though SIB did not revert to originally observed placebo levels during a second placebo phase. An increase in social relatedness also was observed during phases of active drug treatment. Opiate theories of self-injury and the possible interrelationship of self-injury with pituitary-adrenal arousal and with social relatedness are discussed.

Social relatedness and autism: current research, issues, directions.

Social relatedness has recently become a primary focus of investigators in the field of autism. This shift to regarding disturbances in social relatedness as one of the defining manifestations of the disorder marks the movement of research on autistic disorder back to its origins, when Kanner first noted the "social and affective" symptoms of autism as pathognomonic. Currently, social impairment in autism is viewed as more pervasively characteristic of the disorder than any other single symptom. Further, there has been a recent proliferation of research designed to document the nature of social deficit in autism, and whether it is primarily affective, communicative, or cognitive in nature, or involves some combination of these three variables. This review summarizes recent research focusing on social relatedness in autism and discusses the implications of these findings.

Effects of naloxone and naltrexone on self-injury: a double-blind, placebo-controlled analysis.

The effects of naloxone hydrochloride (Narcan) and naltrexone hydrochloride (Trexan) on the pervasive self-injury of a 12-year-old autistic and mentally retarded girl were examined. Using separate multiple schedule (A1/B/B') and withdrawal (A-B-A1B-A1) single-subject experimental designs, we investigated the effects of both opiate antagonists in serial fashion under double-blind, placebo-controlled conditions. Results of the two studies showed that self-injury increased during the naloxone trial, whereas a decrease to near zero rates of self-injury was observed following treatment with naltrexone. The differential effect produced by the two drugs was discussed in terms of drug half-life and the operant conditioning theory of extinction. Follow-up data showing near zero rates of self-injury for 22 months following the conclusion of active treatment with naltrexone indicated that the intervention produced a durable result.

Maternal and paternal alcohol consumption increase offspring susceptibility to Pseudomonas aeruginosa ocular infection.

Male rats that consumed liquid alcohol diets containing 35%, 17.5 or 0% ethanol-derived calories for a minimum of 3 weeks were bred to females which were fed similar diets during pregnancy. At approximately 50 days of age, offspring were challenged with 10 X 10(7) Pseudomonas aeruginosa onto the scarified cornea. The ocular response, evaluated macroscopically, for 3 weeks, revealed a significant dose-related effect of both maternal and paternal alcohol exposure. The higher the parental alcohol consumption the earlier and the more frequently the cornea of progeny perforated. There was no effect of sex of offspring or interaction between maternal and paternal factors. Histopathology confirmed the above data in that progeny of parents receiving 0% or lower-dose alcohol treatment had less severe corneal pathology than progeny of parents with higher alcohol doses.

Depression in mentally retarded children.

Thirty-one mentally retarded emotionally disturbed children, hospitalized within a university medical school's psychiatric intensive care program, were matched on age and sex and compared to 31 children from a normal school setting on depression. Measures included the Child Depression Inventory (CDI) and the Child Behavior Profile (CBP), with children being compared on total and subfactor scores for both measures. Depression and its various subcomponents were more prevalent in the mentally retarded group. There were no significant sex or age differences. Degree of overall psychopathology and depression were highly related. The relationship between criteria for depression on the CDI and CBC were also made. Correlational data showed a strong relationship between the cut-off scores for both measures, an important finding because they were based on norms established with children of normal intelligence. These data suggest that similarities exist between depression in mentally retarded children and those without such cognitive handicaps. The relationship of depression to other forms of psychopathology in the group of 31 emotionally disturbed mentally retarded children was also examined. A wide range of disorders including schizophrenia, aggression, withdrawal, and hyperactivity were evaluated. These are the first empirical data with mentally retarded children in the United States that are aimed specifically at evaluating depression, and should be useful to the clinicians in better understanding the phenomenon.

The assessment of mood and affect in developmentally disabled children and adolescents: the Emotional Disorders Rating Scale.

There is an unmet need for a reliable method of evaluating disorders of mood and affect in developmentally disabled children and adolescents. Such a measure is required for both accurate diagnosis and treatment monitoring in this population. An extensive review of existing assessment techniques confirms that: (a) current techniques for the evaluation of emotional disorders in cognitively normal individuals are inappropriate for most children with developmental disabilities; and (b) current instruments designed for the assessment of developmentally disabled children pay inadequate attention to affective symptoms. In this paper, the preliminary version of a new instrument, the "Emotional Disorders Rating Scale for Developmental Disabilities" (EDRS-DD), designed to evaluate mood and affect in children and adolescents with developmental disabilities, is presented. A pilot study indicates that interrater agreement is good.