Complement-activating immune deposits in systemic lupus erythematosus skin.

Immune deposits at the cutaneous basement membrane zone are a characteristic feature of systemic lupus erythematosus. Previous studies using immunofluorescent methods to detect complement components have provided evidence that some deposits contain immune complexes capable of activating complement. However, this important biologic property of complexes has not been detected or measured using functional assays, and it has not been determined whether immune deposits can activate complement at the basement membrane zone. In this study immune deposits in biopsies of lupus skin have been examined using direct immunofluorescence for the third component of complement (C3) to detect complement deposited in vivo. In addition, the deposits have been studied using the leukocyte attachment assay and indirect C3 binding immunofluorescence to detect and measure complement activation at the basement membrane zone in vitro. The results show that complement activation occurs at the basement membrane in some but not all lupus skin containing immunoglobulin deposits, that deposits differ quantitatively in their ability to activate complement, and that direct C3 immunofluorescence is a relatively insensitive method for detecting complement-activating complexes. The results provide functional evidence suggesting that immune deposits in some lupus skin are complement-activating complexes and potentially capable of activating complement at the basement membrane in vivo. Furthermore, the results suggest functional assays for evaluating complement-activating complexes may be valuable supplements to immunofluorescence in exploring the relationship between immune deposits and systemic and cutaneous disease.

Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

Studies on smaller (approximately 315 microM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules.

UNLABELLED: The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700-1200 microm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (approximately 315 microm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 microm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 +/- 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 microm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-microm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 +/- 8 mmHg and remained elevated after 4 h (12 +/- 3 mmHg), but returned to normal (8 +/- 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. CONCLUSION: intrahepatic implantations of approximately 315-microm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Executing effective road safety advertising: are big production budgets necessary?

Twelve (12) road safety television commercials (TVCs) ranging in production costs from $A15,000 to $A250,000 (current prices) were evaluated using standard advertising pre-test procedures. The twelve ads covered four road safety behaviours (speeding; drink driving; fatigue; and inattention), and included a variety of executional types within and across behaviours. One ad in each of the four behaviours was an expensive TAC and ($A200,000 or more). The testing procedure assessed respondents' self-reported impact of the ad on their future intentions to comply with the road safety behavior advocated in the ad. Just under 1000 appropriately screened motor vehicle drivers license holders were recruited via street intercept methods and randomly allocated to one of the twelve and exposure conditions. The results showed that while the two best performing ads were highly dramatic TAC ads showing graphic crash scenes, these were also the most expensive ads to produce, and, being 60 and 90 s, the most expensive to air. In several cases, 30 s low cost talking heads testimonials performed equally as well as their far more expensive counterparts. We conclude that big production budgets may not be necessary to create effective road safety advertising.

Does deprivation affect breast cancer management?

We evaluated whether social deprivation affected decision-making for breast cancer surgery. Of 3419 patients, 53.6% had mastectomy and this was predicted by deprivation, age, tumour size and hospital, all of which retained significance on multivariate analysis, except deprivation. Pathological characteristics and surgical decision-making determined choice of operation not deprivation.

Young people's response to death threat appeals: do they really feel immortal?

Threat appeals are used frequently in health promotion, with threats of (premature) death common in some areas, e.g. 'quit smoking or you'll die'. There is a common notion that young people feel they are immortal. Accordingly, we investigated whether young people would respond less to threats of death than to non-death threats and whether younger people would respond less to death threats than older people. This study was conducted with smokers in two age groups (16-25 and 40-50 years). Each respondent was exposed to one message about the threat of emphysema, either a death or non-death message. Younger smokers did not respond more to non-death threats than death threats and expressed a higher level of response to all threats than older smokers. It would appear that death threats are effective with young people and so we conclude that they do not feel immortal. An additional finding was that older females responded significantly more to non-death threats than older males. Death threats may not be effective with older females and a segmentation approach may be advisable when targeting older people using death threats in health promotion campaigns.

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravital stain.

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction. Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.

Time course of transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the host reaction to alginate-poly-L-lysine microcapsules following implantations into rat epididymal fat pads.

Microencapsulation of islets of Langerhans within semipermeable membranes has been proposed to prevent their immune destruction after transplantation. However, the successful application of this method is impaired by a pericapsular reaction, which eventually induces graft failure. Our goal is to study the role of cytokines in the pathogenesis of this reaction, using the model of alginate-poly-L-lysine microcapsule implantation into Wistar rat epididymal fat pads (EFP). The specific objective of this study was to determine the time course of transforming growth factor (TGF)-beta(1) mRNA expression by semi-quantitative reverse transcriptase-polymerase chain reaction. Microcapsules induced an increase of TGF-beta(1) mRNA expression that reached a maximum 14 days after implantation. Seven, 14, 30, and 60 days after microcapsule implantation, the expression of TGF-beta(1) mRNA was significantly higher in pericapsular infiltrate cells than in nonimplanted EFP cells (p<0.05, p<0.0001, p<0.005, and p<0.01, respectively). Injection of physiological saline induced a small and gradual augmentation of TGF-beta(1) mRNA expression with a maximum 30 days after injection (p<0.01 vs. nonimplanted EFP cells). These results demonstrated that microcapsule implantation, in comparison with saline injection, induce an early, extended, and amplified TGF-beta(1) mRNA expression. This suggests that TGF-beta(1) plays a role in the pathogenesis of the pericapsular host reaction.

Exploring children's conceptions of smoking addiction.

Tobacco addiction is a major health problem for both adults and young people--between 20 and 60% of adolescents are dependent on nicotine and more than two-thirds who attempt to quit experience withdrawal symptoms. Yet, anti-smoking efforts targeted at children emphasize primary prevention and ignore addiction education, which is generally considered relevant only to adult smoking and cessation efforts. This study reports children's concepts of smoking and addiction from interviews with 32 Western Australian boys and girls, aged 9-10 years, all non-smokers. Results suggest children's concepts of smoking addiction may influence attitudes toward smoking trials. Children who thought addiction happened immediately were committed to never smoke at all. Others who thought addiction happened after several cigarettes expressed intentions to experiment. These children believed they could avoid addiction by smoking less than the number of cigarettes that caused addiction. Children who defined addiction as 'liking' or 'enjoying the taste of cigarettes' believed they could avoid being 'hooked' as long as they managed to avoid enjoying the experience. Recent findings that children can get 'hooked' within a few days of smoking highlight the importance of addiction strategies targeted at children, particularly in relation to influencing intentions to experiment with smoking.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. V. Determination of carbohydrate and protein permeation through microcapsules by reverse-size exclusion chromatography.

Membrane molecular weight (MW) cut-off is a critical factor for immunoprotection of transplanted microencapsulated cells as well as for graft survival. Our goal was to study dextran and protein permeation through small (<350 microm in diameter) alginate-poly-L-lysine microcapsules made with an electrostatic system. Microcapsules were packed into a column, and gel-sieving chromatography was performed with proteins and dextrans of known MW. The objectives of this study were (1) to validate this approach for the assessment of the MW cut-off of <350 microm-in-diameter microcapsules and (2) to evaluate the effect on MW cut-off of changes in experimental conditions. Elution profiles of proteins suggest that the MW cut-off of our small microcapsules lies between 14,500 and 44,000 Da whereas dextrans > or =19,000 Da were excluded. The increase in poly-L-lysine (PLL) concentration from 0.02 to 0.08% reduced the MW cut-off. Increasing the PLL MW from 11.6 to 69.6 kDa induced no change in the MW cut-off. The results also show that the method can be used to discriminate between adsorption and absorption and that insulin diffuses freely across the microcapsule membrane. This method will be useful in establishing the ideal MW cut-off, in optimizing microcapsule characteristics, and in performing routine quality controls.

Replication of clinical innovations in multiple medical practices.

BACKGROUND: Many clinical innovations had been successfully developed and piloted in individual medical practice units of Kaiser Permanente in North Carolina during 1995 and 1996. Difficulty in replicating these clinical innovations consistently throughout all 21 medical practice units led to development of the interdisciplinary Clinical Innovation Implementation Team, which was formed by using existing resources from various departments across the region. REPLICATION MODEL: Based on a model of transfer of best practices, the implementation team developed a process and tools (master schedule and activity matrix) to quickly replicate successful pilot projects throughout all medical practice units. The process involved the following steps: identifying a practice and delineating its characteristics and measures (source identification); identifying a team to receive the (new) practice; piloting the practice; and standardizing, including the incorporation of learnings. The model includes the following components for each innovation: sending and receiving teams, an innovation coordinator role, an innovation expert role, a location expert role, a master schedule, and a project activity matrix. Communication depended on a partnership among the location experts (local knowledge and credibility), the innovation coordinator (process expertise), and the innovation experts (content expertise). RESULTS: Results after 12 months of working with the 21 medical practice units include integration of diabetes care team services into the practices, training of more than 120 providers in the use of personal computers and an icon-based clinical information system, and integration of a planwide self-care program into the medical practices--all with measurable improved outcomes. CONCLUSION: The model for sequential replication and the implementation team structure and function should be successful in other organizational settings.

Phasic and tonic reflexes evoked in human antagonistic wrist muscles by tendon vibration.

The electromyographic reflex responses of the voluntarily contracting wrist flexor and extensor muscles to periods of vibration-evoked enhanced, Ia-dominated afferent discharge from flexor carpi radialis (FCR) were studied in normal human subjects. Three main response phases were characterised, namely, (i) phasic 'on' responses elicited at the commencement of stimulation, (ii) tonic response levels occurring during prolonged stimulation and (iii) phasic 'off' responses elicited at the termination of stimulation. The phasic 'on' reflex responses of FCR and extensor carpi radialis (ECR) comprised, respectively, a peak of autogenetic excitation of group mean latency 18.8 ms and a trough of reciprocal inhibition of group mean latency 38.0 ms. Prolonged (2 s) trains of FCR (agonist) vibration evoked a phase of tonic reflex excitation in FCR whose mean level was significantly increased, by 20%, above pre-stimulus activity and which did not change over the 0.5-2.0 s vibration period. Progressive reduction of the duration (from 2000 ms to 100 ms) of vibration trains demonstrated that phasic disfacilitatory 'off' troughs regularly occurred, with a consistent latency (mean 24.2 ms), on withdrawal of each period of enhanced Ia-input. This indicates that the responsible excitatory reflex mechanism was operational for the entire duration of each of the vibration periods tested. The extra latency (on average 5.4 ms) of phasic 'off' relative to 'on' responses may be attributed to factors (e.g. 5-10 ms duration of unitary muscle action potentials and afterdischarge in reflex pathways) which inevitably delay the appearance of overt disfacilitatory reductions in EMG rather than the involvement of different reflex pathways. Thus, short-latency, possibly monosynaptic, reflex excitation contributed throughout the entire tonic excitatory response. Sustained FCR (antagonist) vibration produced a significant tonic reciprocal inhibitory reflex depression, by 7% pre-stimulus EMG, of ECR activity which remained steady during the 0.5-2.0 s vibration period. The absence of well-defined phasic disinhibitory 'off' responses in ECR suggests that the contribution of oligosynaptic reflex inhibitory mechanisms to the tonic suppression of activity occurring during continuing vibration is relatively small.

Alginate-poly-L-lysine microcapsule biocompatibility: a novel RT-PCR method for cytokine gene expression analysis in pericapsular infiltrates.

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.