Parent and Teacher Perspectives on Factors Decreasing Participation in School-Based Vision Programs.

Purpose: To examine factors decreasing participation in school-based vision programs from parent and teacher perspectives.Methods: We conducted 41 semi-structured focus groups (20 parent groups, 21 teacher/staff groups), at 10 Baltimore and 11 Chicago public elementary and middle schools offering school-based vision programs. School-based vision programs provided vision screening, eye exams, and eyeglasses if needed. Focus groups ranged in size from 2-9 participants (median = 5). Sessions were recorded, transcribed, and coded through an iterative process to develop themes using inductive analysis.Results: Ninety parents and 117 teachers/staff participated. Participants identified five major factors decreasing participation in school-based vision programs: (1) challenges with the consent form, including distribution, collection, and literacy and language barriers; (2) having existing eye care; (3) misunderstandings about the program, especially related to cost and insurance; (4) difficulty raising parental awareness of the program; and (5) certain attitudes towards vision, eye care, and school-based programs, including low prioritization of eye care, mistrust of the program, fear of sharing private information, not believing their child needs glasses, and reluctance accepting 'subsidized' services.Conclusion: Parents and teachers identified important structural barriers to participation (i.e., consent form challenges and low parental awareness) and specific reasons for non-participation (i.e., attitudes, misunderstanding of the program, existing eye care) in school-based vision programs. Effective strategies are needed to facilitate return of consent forms and promote awareness of school-based vision programs among parents. Programs should also target services towards those currently without access to eye care and increase awareness about paediatric vision needs.

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.

We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.

Modification of the stability of phospholipase A2 by charge engineering.

Electrostatic interactions play an important role in stabilizing the folded conformation of globular proteins. Here we predict the change in stability of charge engineered mutants, construct these mutants and compare the predicted change in stability with that observed. The change in stability was correctly predicted for two of the three mutants and the factors responsible for the discrepancy between observation and prediction for the third mutant are discussed.

Differences in dermal analogs influence subsequent pigmentation, epidermal differentiation, basement membrane, and rete ridge formation of transplanted composite skin grafts.

This study evaluated the in vitro and in vivo function of composite skin equivalents based on two different dermal analogs. Keratinocytes derived from the same dark-skinned neonatal foreskins were seeded onto both acellular human dermis and fibroblast-contracted collagen gels. Each type of composite graft readily formed an epithelium in vitro. However, the undulating surface of the acellular dermis acted as a template and organized the seeded keratinocytes into a rete ridge-like pattern, whereas the smooth surface of the fibroblast-contracted collagen gels generated an epithelium with a linear basal layer. Moreover, when acellular dermis was used, the composite grafts demonstrated enhanced melanocyte proliferation. When transplanted to athymic mice, both composite grafts formed a fully differentiated human epidermis, but repigmentation of the grafts when acellular dermis was used was more extensive and only the epidermis on the fibroblast-contracted collagen gels showed signs of hyperproliferation at 6 weeks after grafting. These results demonstrate that the type of dermal analog incorporated into a composite skin graft can influence the subsequent functionality of the skin substitute.

Time of transforming growth factor beta 1 inoculation alters the incubation of BSE in mice.

Groups of 20 C57BL/6J mice (10 males and 10 females) were given BSE strain 301C i.p. and subsequently given 2 microg recombinant human TGFbeta1 s.c. at single or multiple times. There was a significant positive correlation between the day of TGFbeta1 administration and incubation time; the later TGFbeta1 was administered after BSE inoculation the longer the incubation time became. The administration of TGFbeta1 at any time point did not significantly alter the distribution or severity of pathology. The effects of TGFbeta1 on BSE pathogenesis appears to be dependent upon its time of administration; early administration shortens the incubation time and late administration lengthens the incubation time.

Clusterin shortens the incubation and alters the histopathology of bovine spongiform encephalopathy in mice.

Clusterin accumulates in significant quantity in prion protein lesions associated with bovine spongiform encephalopathy (BSE) and we therefore sought to elucidate its ability to alter BSE pathogenesis and incubation time by comparison of wild type C57BL/6J mice and clusterin knock out (ko) mice. The ko mice had a 40 day increase in mean incubation time compared to wild type mice. PrP deposition in the medulla was less aggregated in clusterin knock out mice when compared to wild type BSE infected mice and a more marked astrocytosis, as determined by GFAP staining, was evident. The vacuolation profiles did not differ between the two strains of mice. Taken together these results suggest that clusterin alters the extracellular deposition of PrP(BSE) and accelerates BSE pathogenesis.

Impaired motor coordination on static rods in BSE-infected mice.

Scrapie and bovine spongiform encephalopathy (BSE) are both progressive neurodegenerative diseases that are transmissible to mice. The onset of clinical symptoms is more subtle and variable in murine BSE than in murine scrapie. Assessment of behavioural changes that occur throughout disease would aid early diagnosis of disease so that more consistent end points could be made and potential therapies could be investigated. C57BL/6J mice inoculated via the intraperitoneal route with 301C BSE or control inoculum were monitored on a fortnightly basis. The end point was when a mouse showed clinical signs as opposed to behavioural signs of BSE for two consecutive observations. Significant loss of motor function, as assessed by mice balancing on a static rod, was observed consistently from approximately 40 days prior to death. No significant differences in home cage activity (locomotion, rearing) or cognitive function (T-maze alternation) were observed. However, there was an increase in digging by BSE-infected mice from an early stage. This data will aid the standardisation of behavioural tests to characterise and assess the onset of BSE.

Analysis of variation of bovine viral diarrhoea virus E2 sequence following transplacental infection of cattle.

The genetic and antigenic diversity observed in field isolates of bovine viral diarrhoea virus (BVDV) is thought to occur during acute infection because of the genetic stability observed in BVDV throughout the lifetime of persistently infected (PI) cattle. In this study, 15 cows in early pregnancy were inoculated with identical challenge doses obtained from a single infectious inoculum of the virologically cloned isolate Pe515nc. In order to examine the diversity that may develop in utero in the PI foetus, the variable E2 sequence of the virus isolated directly from the serum of each PI calf was compared. A high degree of sequence similarity was demonstrated, with 0-4 nucleotide differences out of 608 bases compared. Thus, the virus showed relatively few genomic changes in any of the PI calves, although we observed that the in utero environment did provide some opportunity for genetic variation to become established.

Co-administration of IL-2 enhances antigen-specific immune responses following vaccination with DNA encoding the glycoprotein E2 of bovine viral diarrhoea virus.

Induction of effective immunity requires the delivery of a protective antigen with appropriate co-stimulatory signals. For bovine viral diarrhoea virus (BVDV) this antigen is the major viral glycoprotein E2. Neutralising antibodies are directed towards the E2 protein and passive transfer of antibodies in serum or colostrum can completely protect against viral infection. DNA vaccination of mice with a construct encoding the E2 glycoprotein induced neutralising antibody levels that were potentially sufficient to prevent virus replication in a challenge system. The co-delivery of interleukin-2 (IL-2) further enhanced the levels of antibody raised. The strong IgG2a component of the antigen-specific antibody suggests a Th1 bias to the immune response induced following vaccination.

Evidence for the presence of two bovine lentiviruses in the cattle population of Bali.

Antibodies directed against two bovine lentiviruses, Jembrana disease virus (JDV) and bovine immunodeficiency virus (BIV), were detected in Balinese cattle sera using two new enzyme-linked immunosorbent assays (ELISAs) based on the combination of capsid (CA) protein and transmembrane (TM) peptides derived from JDV or BIV sequences. Twenty eight of the 77 sera tested on the JDV ELISA showed anti-JDV antibodies with an unequal distribution of seropositive animals throughout the different districts of Bali. Furthermore, when 17 of the JDV positive sera were tested on Western blot, using the same JDV CA antigen, only 13 were judged positive confirming that the ELISA was a more sensitive technique for the detection of seropositive animals. Finally, 9 of the 49 JDV seronegative animals showed anti-BIV antibodies when tested on BIV-specific ELISA. These two ELISAs appeared to be highly sensitive for the detection of anti-JDV and anti-BIV antibodies. Moreover, for the first time, animals showing antibodies against BIV were identified on the main island of Bali and on the JDV-free Nusa Penida island.

Cloning of equine chemokines eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, mRNA expression in tissues and induction by IL-4 in dermal fibroblasts.

We report the cloning of four equine CC chemokines, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, which show high levels of identity with their respective homologous sequences in other species. Using a multiplex RT-PCR, we have studied the constitutive mRNA expression of these four CC chemokines in skin, lung, liver, spleen, jejunum, colon and kidney of normal adult horses and compared this data with the eosinophil counts in the same samples. We demonstrate that eotaxin mRNA is only expressed in jejunum and colon, where there are large numbers of eosinophils suggesting that eotaxin might be recruiting eosinophils in the normal digestive tract of the horse. MCP-1 and MCP-4 are expressed in all tissues whereas MCP-2 is only found in some samples of lung, spleen, liver and kidney. We also report the early induction (2h) of equine eotaxin and MCP-4, and the up-regulation of MCP-1 by interleukin-4 in dermal fibroblasts, suggesting these chemokines might be involved in equine skin allergic diseases.

Characteristics predicting successful outcomes of participants with severe mental illness in supported education.

OBJECTIVE: The study sought to identify characteristics of participants in a supported education program that were related to a successful outcome. Supported education programs provide rehabilitation and support services to help people attain postsecondary education. METHODS: A total of 147 persons who completed such a program were interviewed six or 12 months later, or at both times, to determine whether they were involved in productive activity, which was defined as engaging in either college or vocational education or in paid employment. Variables examined as predictors of productive activity were demographic characteristics; education and work background; social support; self-perceptions related to self-esteem, empowerment, quality of life, and school self-efficacy; and illness-related variables, including diagnosis, symptoms, and length of illness. RESULTS: Multivariate logistic regression identified the strongest predictor as productive activity at baseline. Marital status was the only significant demographic variable in the model; single participants were less likely to be engaged in productive activity. For participants who reported more frequent contact with their social network, the likelihood of engagement in productive activity was higher, and for those who reported more encouragement for education from their network, the likelihood was lower. A lower level of adjustment in the financial domain decreased the likelihood of productive activity, and a higher level of problems with housework increased the likelihood. No illness-related variable or self-perception was a significant predictor. CONCLUSIONS: Factors related to a successful outcome from a supported education program for persons with severe mental illness are also likely to be important factors for nondisabled populations. Among those with mental illness, social support is a key factor in attaining educational and vocational goals.

An inducible gene expression system for Neurospora crassa.

Neurospora crassa acetyl CoA synthetase is highly induced when the growing mycelium is transferred from sucrose- to acetate-based medium. The inducible promoter of this gene has been isolated and used to control the expression of glutamate dehydrogenase. Transformants containing this expression cassette show gdh levels up to 25 times higher than the nontransformed host strain. This expression cassette will form the basis of a system of heterologous gene expression.

A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos.

Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

Consumers as mental health providers: first-person accounts of benefits and limitations.

Community support programs are increasingly establishing paid service positions designated exclusively for consumers. Project WINS (Work Incentives and Needs Study), a hybrid case management-vocational program for individuals with severe mental illness, used consumers as peer support specialists (PSSs) to supplement professional roles. Semistructured interviews were conducted with PSSs about 12 months after their employment ended. They identified substantial personal benefits specific to consumer-designated roles (e.g., a "safe" employment setting with accommodations) and general benefits from employment. Problems described were just as numerous, encompassing attitudes toward assigned peers and costs to their own well-being. Critical commentary addressed program operations (structure, supervision, and training needs) and problems in the mental health system. The authors discuss the changed sense of self that service provider roles can create for consumers and suggest that mental health administrators provide anticipatory socialization for this service innovation throughout their agencies and ongoing supports for consumers in their new roles.

Role of the chemokine eotaxin in the pathogenesis of equine sweet itch.

The chemokine eotaxin is involved in the recruitment of eosinophils and T helper 2 lymphocytes in human allergic diseases, and drugs that block its activity, including eotaxin receptor (CCR3) antagonists, are being developed. The authors have recently cloned the horse ortholog of eotaxin and shown that it can induce equine eosinophil migration and activation in vitro. Moreover, eotaxin mRNA expression was upregulated in cultured horse dermal fibroblasts exposed to equine interleukin-4, suggesting a possible source of this eosinophil chemoattractant in equine skin. The results of this study show that eotaxin and monocyte chemoattractant protein (MCP) 1, but not MCP-2 or MCP-4, mRNA expression is upregulated in skin biopsies of sweet itch lesions when eosinophils are present, when compared with clinically normal skin from the same ponies.