A complex flow pattern of low shear stress and flow reversal promotes monocyte binding to endothelial cells.

Predilection sites for atherosclerosis within the vasculature are characterized by low shear stress and flow reversal. In this study, endothelial cells were exposed to a complex flow pattern that was characterized by particle velocity determination. Bovine aortic endothelial cells exposed to low shear stress and flow reversal demonstrated higher levels of monocyte binding compared to endothelial cells exposed to one-directional flow. In addition, endothelial cells exposed to low shear stress and flow reversal responded to inflammatory stimuli with substantial increases in monocyte binding, similar to that seen in cells exposed to one-directional flow. These findings suggest a mechanism by which areas of low shear stress and flow reversal are predisposed to the development of atherosclerotic lesions.

Enhanced protective antibody responses to PspA after intranasal or subcutaneous injections of PspA genetically fused to granulocyte-macrophage colony-stimulating factor or interleukin-2.

Antibody to pneumococcal surface protein A (PspA) has been shown to be protective for Streptococcus pneumoniae infections in mice. In an attempt to define a model for inducing protective antibody to PspA in the absence of adjuvant, we designed two genetic fusions, PspA-interleukin-2 [IL-2]) and PspA-granulocyte-macrophage colony-stimulating factor (GM-CSF). These constructs maintained high cytokine function in vitro, as tested by their activity on IL-2 or GM-CSF-dependent cell lines. While intranasal immunization with PspA induced no detectable anti-PspA response, both PspA-IL-2 and PspA-GM-CSF stimulated high immunoglobulin G1 (IgG1) antibody responses. Interestingly, only the PspA-IL-2, not the PspA-GM-CSF, construct stimulated IgG2a antibody responses, suggesting that this construct directed the response along a TH1-dependent pathway. Comparable enhancement of the anti-PspA response with similar isotype profiles was observed after subcutaneous immunization as well. The enhancement observed with PspA-IL-2 was dependent on IL-2 activity in that it was not seen in IL-2 receptor knockout mice, while PspA in alum induced high-titer antibody in these mice. The antibody was tested for its protective activity in a mouse lethality model using S. pneumoniae WU-R2. Passive transfer of 1:90 dilutions of sera from mice immunized with PspA-IL-2 and PspA-GM-CSF elicited protection of CBA/N mice against intravenous challenge with over 170 50% lethal doses of capsular type 3 strain WU2. Only 0.17 microg or less of IgG antibody to PspA was able to provide passive protection against otherwise fatal challenge with S. pneumoniae. The data demonstrate that designing protein-cytokine fusions may be a useful approach for mucosal immunization and can induce high-titer systemic protective antibody responses.

Bacterial lipoproteins may substitute for cytokines in the humoral immune response to T cell-independent type II antigens.

Bacterial lipoproteins share a common structural motif that has been shown to stimulate proliferation and Ig secretion of murine B cells, in a manner distinct from that mediated by LPSs. Studies of lipoprotein-mediated B cell activation utilized heterogeneous populations of lymphoid cells, leaving unresolved their ability to directly activate resting B cells, as well as their ability to interact with other B cell stimuli. Using highly enriched and/or sort-purified resting murine B cells, we demonstrate that, in contrast to previous reports, lipoproteins (lipoprotein-D, lipoprotein-OspA, and/or the synthetic analogue Pam3Cys) stimulate little, if any, proliferation or Ig secretion in resting B cells. However, when combined with a multivalent membrane (m)Ig-mediated cross-linking signal, dextran-conjugated anti-IgD Abs (alpha delta-dex), lipoproteins mediate up to 10,000-fold inductions in IgM secretion and up to 25-fold enhancements in cellular proliferation relative to that observed with alpha delta-dex alone, in the absence of added cytokines. This mIg-mediated enhancement of Ig secretion was not observed when B cells were stimulated with bivalent, unconjugated anti-Ig. CD40 ligand (CD40L), shows a similar, although somewhat more moderate, synergy with lipoproteins for induction of proliferation and IgM secretion. By contrast, lipoproteins by themselves are relatively ineffective at costimulating Ig secretion in the presence of various combinations of cytokines. These data suggest that bacteria may induce Ag-specific humoral immunity through the action of bacterial polysaccharides that mediate an Ag-specific multivalent mIg signal, in concert with bacterial lipoproteins that deliver ancillary signals, without a requirement for recruitment of non-B cell types.

Mechanical stimulation induces intercellular calcium signaling in bovine aortic endothelial cells.

To study the mechanism by which endothelial cells respond to mechanical forces, we used digital fluorescence microscopy to measure changes in intracellular Ca2+ concentration ([Ca2+]i) in primary cultures of bovine aortic endothelial cells in response to mechanical stimulation. Before stimulation, [Ca2+]i was stable (approximately 50-75 nM). When an individual cell within the monolayer was mechanically stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and spread in the form of a wave from the site of contact to the cell edges. After a delay of approximately 1 s, nonstimulated adjacent cells showed a similar spreading rise in [Ca2+]i. This outwardly radiating [Ca2+]i wave involved progressively more distal cells to a radius of 4-6 cells. The time delay before the wave appeared in adjacent cells increased, and peak [Ca2+]i in each cell decreased with distance from the stimulated cell. In the absence of extracellular Ca2+, there was no increase in [Ca2+]i in the stimulated cell, yet a wave of increased [Ca2+]i occurred in neighboring cells as if communicated from the stimulated cell. These results indicate that endothelial cell mechanosensitivity results in increases in [Ca2+]i and that the temporospatial dynamics of intercellular Ca2+ signaling are mediated by a diffusible substance other than Ca2+.

Ocular and vision defects in preschool children.

Ocular and/or vision defects are one of the commonest reasons for the referral of young children to hospital. In a survey of a birth cohort in one health district, 7.1% of children were diagnosed as having such defects by their fifth birthday; 2.1% were detected before the age of 2 years, and 5.1% between 2 and 5 years. Up to the age of 2 years, low birthweight children and those who require postnatal special care had a higher risk of having an ocular or vision defect diagnosed and were more likely to have serious visual impairment than other children. In contrast, between the ages of 2 and 5 years of age these high risk children showed no continuing increased risk of having a defect diagnosed, nor did they show any differences in the severity or type of vision defects compared with other children. Averaged over the years studied, the incidence of defects presenting to specialist eye clinics among all 2-5 year olds was 1.7%, higher than the 1.1% found for 0-2 year olds. This increase consisted primarily of children with refractive errors only.

Bone morphogenetic protein expression in human atherosclerotic lesions.

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.

Orthoptists reduce false-positive hospital referrals.

This paper describes changes in the pattern of new referrals of children from birth to 2 years-of-age to the Hospital Eye Service in Oxford, following the implementation of a community-based orthoptic secondary vision-screening programme. The findings show that the number of false positive referrals was reduced by a half following the introduction of the service; false-positive referrals were also discharged sooner. This paper reports the findings of this survey and the results demonstrate the cost-effectiveness of the programme compared to outpatient attendance.

Ocular and visual defects in a geographically defined population of 2-year-old children.

Ocular and/or visual defects were diagnosed by age 2 years in 2.1% of infants born in 1984 in one health district. Infants of low birth weight or infants requiring special care in the neonatal period had a two and a half times greater risk of vision and ocular defects than the remainder of the population. In particular, the rate of squint and neurological disease affecting the visual system was significantly greater in this high risk group than in the low risk group. The degree of visual impairment in the high risk group tended to be more severe than in the low risk group. Overall, however, the majority of children with vision or ocular defects (85%) were in the low risk group.

Vision screening at 8 and 18 months. Steering Committee of Oxford Region Child Development Project.

OBJECTIVE: To determine the effectiveness of an existing screening programme based in the community for ocular and vision defects in infants considered at increased risk of such defects. DESIGN: Children with ocular or vision defect by the age of 2 were ascertained by searching records. Those from populations at high risk were matched with their results from screening tests. The characteristics of the cases among this population were compared with those of the cases in the remainder of the population. Patterns of referral and age at referral were studied in both groups. SETTING: The study was conducted within Oxfordshire Health District. SUBJECTS: 433 Children at high risk born in 1984 to mothers living in the health district at delivery and who either weighed less than 2000 g or weighed 2000 g and over and required admission to a special care nursery for longer than 24 hours. The low risk population (6254) were infants without these characteristics who were resident in the health district at the time of referral. INTERVENTIONS: Screening tests for vision or ocular defects already routinely used were applied by health visitors at 8 and 18 months to the children at high risk. MAIN OUTCOME MEASURE: Comparison of results of screening tests with vision and ocular defects detected by the age of 2. RESULTS: Screening tests in current use for vision loss and squint in this age group were insensitive and had a low positive predictive value when applied to a high risk population. Defects that were not apparent on direct inspection were unlikely to be detected by these tests. In the high risk group the relative risk of having a defect was 2.8 (95% confidence interval 1.8 to 4.5) but 85% of all cases detected by the age of 2 were in children at low risk. Referral patterns and age of referral differed in the two groups. CONCLUSIONS: Screening by health visitors of high risk populations contributes little to the detection of vision and ocular defects. This type of evaluation needs to be applied also to low risk populations, who have different referral patterns and contribute most of the cases.