Enhancement of tumor-specific immune response with plasmid DNA replicon vectors.

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.

Single center experience of a new intensive induction therapy for ewing's family of tumors: feasibility, toxicity, and stem cell mobilization properties.

PURPOSE: To examine the feasibility, tolerability, and toxicity of an intensified induction regimen (vincristine, ifosfamide, doxorubicin, and etoposide [VIDE]) in patients with newly diagnosed Ewing's family of tumors (EFT); to assess ability to maintain dose-intensity, and predictability of peripheral-blood stem cell mobilization. PATIENTS AND METHODS: Thirty patients were treated with vincristine 1.4 mg/m2 (maximum 2 mg) on day 1, doxorubicin 20 mg/m2, ifosfamide 3 g/m2 plus mesna and etoposide 150 mg/m2 on days 1 to 3. Cycles were given every 21 days for up to six cycles. RESULTS: One-hundred and seventy cycles of VIDE were given. The median treatment interval was 21 days (21 to 42) and nadir count: hemoglobin 8.3 (6.3 to 11.9), neutrophils 0.045 (0.0 to 2.1), and platelets 45 (3 to 343). There were 96 episodes of infection requiring hospitalization (56%). Growth factor support reduced infectious complications by 34%. Etoposide dose was reduced, or omitted, in 24% of cycles. Four patients did not complete six cycles due to unacceptable toxicity and one patient progressed on treatment. Twenty patients underwent peripheral-blood stem cell harvesting, 15 after cycle 3, and five after cycle 4. Median CD34+ yield was 4.6 x 106/kg per patient (1.8 to 14.5). Overall response to treatment, measured in 24 patients, was 88%. Seven of 11 patients undergoing surgery achieved greater than 90% necrosis of tumor (64%). CONCLUSION: VIDE is an effective induction regimen with substantial but acceptable toxicity that allows predictable mobilization of stem cells. Maintenance of dose-intensity is feasible in the majority of patients. Growth factors play a role in maintaining dose-intensity and reduce infectious complications.

Teleophthalmology for first nations clients at risk of diabetic retinopathy: a mixed methods evaluation.

BACKGROUND: Access to health services is a particular challenge for First Nations (aboriginal Canadians) communities living in remote or underserviced areas. Teleophthalmology can provide them with the same level of retinal screening services provided to those in urban centers. This screening can lead to the identification of high-risk individuals who can then be monitored and receive treatment related to their diabetes or other health issues. OBJECTIVE: The intent was to develop, implement, and evaluate a service delivery model for teleophthalmology screening and follow-up for at-risk and diabetic First Nations clients on Vancouver Island, British Columbia, Canada. METHODS: A highly consultative, culturally appropriate, and collaborative approach was used to develop and deploy a teleophthalmology service delivery model to First Nations communities. This project was evaluated with regard to utilization and operational costs. Also, clinicians and team members involved in the teleophthalmology project provided assessments of the teleopthalmology quality, productivity, and access. Health providers in First Nations communities provided their perceptions of areas of improvement for the remote retinal screening services, areas where expansion of services could be offered, and opportunities to increase client education and health promotion. RESULTS: All 51 First Nations communities on Vancouver Island expressed interest in receiving teleopthalmology services. During the 1-year project, teleopthalmology clinics were held in 43 of 51 communities on Vancouver Island. During these clinics, 524 clients were screened and 140 of those clients were referred to a general ophthalmologist, family doctor, retinal specialist, optometrist, or other provider. Ratings of teleopthalmology system quality, information quality, service quality, and system usage were positive. Satisfaction with the teleopthalmology project was high among clinicians involved with the project. Satisfaction was also high among health providers in First Nations communities, with clinic scheduling identified as a potential area of improvement moving forward. The average cost savings per client, taking project costs into consideration, was calculated to be CAN $28.16, which was largely due to the elimination of client travel costs. CONCLUSIONS: Teleophthalmology was a welcome addition to health services by the First Nations communities on Vancouver Island, as evidenced by the 100% rate of interest from those communities. There was no evidence of dissatisfaction by clinicians involved in the teleopthalmology project or by First Nations community health providers. The now-operational teleopthalmology program is a testament to the early success of the project.

Antigen-specific immunity. Th cell-dependent B cell responses.

Helper T cell-regulated B cell responses constitute a major component of the primary immune response to many pathogens. The subsequent development of antigen-specific immune memory is one critical outcome of this primary adaptive immune response. Antigen-specific immunity develops through a series of intercellular information exchanges organized around cognate T cell receptor-peptide/MHC interactions. Here, we discuss these complex molecularevents andtheircellularconsequences in a serial synapsis model of adaptive immunity. Our laboratory has developed strategies to isolate antigen-specific Th cells and B cells to analyze gene expression and cellular function in single responding lymphocytes directly ex vivo. These studies provide insight into the regulation and cellular organization of antigen-specific immune responses in vivo.

Catecholamines during cardiopulmonary resuscitation for cardiac arrest.

Serum catecholamines were measured during continued prolonged cardiopulmonary resuscitation and after 10 mg increments of intravenous epinephrine. This was part of an ongoing trial of 10 mg epinephrine versus placebo. Eight patients were in the placebo arm and seven in the epinephrine arm and the rhythms were two ventricular fibrillation, nine asystole and four electromechanical dissociation. Data were analysed by time from onset of the cardiac arrest and samples were analysed for levels of DHPG (dihydroxyphenylglycol) nor-epinephrine, epinephrine, DOPA (dihydroxyphenylalanine) and DOPAC (dihydroxyphenyl acetic acid). There was a significant (P < 0.001) difference between arterial and venous samples of epinephrine but not the other catecholamines. High levels of catecholamines were maintained in all time phases except for nor-epinephrine where significant (P < 0.0003) reduction occurred progressively after 20 min. Non-steady state kinetics were suggested between epinephrine and nor-epinephrine and DHPG and nor-epinephrine for the first 20 min. Very large increases in epinephrine were achieved with administered 10 mg epinephrine and this resulted in high DHPG levels supporting the experimental belief that exogenously administered epinephrine induces myocardial release of nor-epinephrine. This data supports the known effects of CPR on catecholamine release. It provides data on the other neurotransmitter hormones and supports the relationships shown in other animal and human data. It is suggested that supplementation with epinephrine during CPR may be unnecessary and the levels reached may be deleterious. Nor-adrenaline supplementation may be necessary after prolonged CPR.

Plasmid DNA-based alphavirus expression vectors for nucleic acid immunization.

Alphavirus-derived vectors are being developed for vaccine, gene therapy and recombinant protein production applications, based in part on observations of transient, high level expression of heterologous genes in eukaryotic cells. Efficient means for launching the RNA alphavirus genome from RNA polymerase II expression cassettes have been developed, obviating the need for transcription in vitro of long cDNA templates. One system being developed from this technology is a layered plasmid DNA vector which, when inoculated directly into animal muscle, launches a self-amplifying alphavirus vector, resulting in subsequent induction of comparatively robust immune responses specific for the expressed antigen.

A profile of grandparents raising grandchildren in the United States.

This article examines the prevalence of grandparent caregiving in the U.S. and presents a national profile of grandparent caregivers based on current data from the national Survey of Families and Households. More than one in ten grandparents are found to have cared for a grandchild for at least 6 months, with most of these having engaged in a far longer-term commitment. Although custodial grandparenting cuts across gender, class, and ethnic lines, single women, African Americans, and low income persons are disproportionately represented. Multivariate logistic analysis indicates that three groups--women, recently bereaved parents, and African Americans--have approximately twice the odds of becoming caregiving grandparents. Implications for further research, policy, and practice are discussed.

Community interventions to support grandparent caregivers.

Community interventions and service programs for grandparents raising grandchildren are being identified and tracked through the Brookdale Grandparent Caregiver Information Project. Based on the first year's examination of 124 such programs, an overview of community intervention effort is provided, with special attention to support groups and comprehensive multi-service programs for grandparent caregivers. Lack of funding and institutional support, and the consequent inability to provide child care, were among key obstacles faced, while sponsorship by health and social service agencies often played a vital role in providing in-kind support and part-time professional staff.

Virtual simulation in palliative lung radiotherapy.

AIMS: To study the accuracy of tumour-volume localisation in a comparison of conventional and virtual simulation for palliative lung radiotherapy. To assess if three-dimensional tumour outlining is necessary for the virtual simulation process. MATERIALS AND METHODS: Ten consecutive patients with non-small cell lung cancer underwent target localisation for palliative lung radiotherapy using conventional and virtual simulation. The treatment fields were initially marked with a conventional simulator using fluoroscopy, plain X-ray film and available diagnostic imaging. Each patient then had a computed tomography (CT), and these simulated treatment fields were reproduced within the virtual simulation planning system. Two clinicians then independently defined treatment fields using virtual simulation alone. The virtual simulation was achieved without outlining the tumour in three dimensions. The coverage of an 'ideal' CT-defined planning-target volume (PTV) was then calculated for each of the virtually and conventionally simulated fields. In addition, the amount of irradiated normal lung was measured using dose-volume histograms (DVH). Field sizes and differences in tumour volume coverage were compared. RESULTS: There was significantly greater tumour volume coverage using virtual simulation compared with conventional simulation (P < 0.03). This advantage was more pronounced in tumours that were larger and those that were closer to the patient's midline. There was no statistically significant difference in the volume of uninvolved lung irradiated between the two methods. CONCLUSION: In this small sample of patients, we have demonstrated improved tumour volume coverage using virtual simulation, without increasing the volume of uninvolved lung treated. A simple but consistent method of virtual simulation for this patient group is offered as an alternative to both PTV-defined CT simulation and fluoroscopy-based conventional simulation.

Alphavirus DNA and particle replicons for vaccines and gene therapy.

Alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. Alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification of the vector RNA. We have developed plasmid DNA and recombinant vector particle delivery systems derived from the prototype alphavirus, SIN. Each system uses RNA polymerase II-based expression of alphavirus genome components and both vector formats are highly efficacious towards inducing robust antigen-specific immune responses in vaccinated animals. To increase the potency of SIN vector particles, which are not known to be lymphotropic, the tropism was re-directed for efficient infection of dendritic cells, both in vitro and in vivo.

PET versus SPECT for determining myocardial tissue viability using fluorine-18-fluorodeoxyglucose.

The U.S. Food and Drug Administration regulates the manufacture and distribution of 18F-FDG. Acquisition and use of this radiopharmaceutical for myocardial imaging at our institution required approval by our Institutional Review Board. Our initial objective was to present a scientific validation for use of this radiopharmaceutical in adjudicating the "viable versus scarred myocardium question," using either a PET scanner or a gamma camera equipped with 511-keV collimators. We believe that our nuclear medicine colleagues who find themselves in a similar business/legal situation regarding the acquisition and use of 18F-FDG will find this review useful. We reviewed the literature to document the efficacy of 18F-FDG, in general, in assessing myocardial viability. We then did a literature-based comparison of PET scanner methodology versus modified gamma camera methodology for imaging 18F-FDG emissions. Imaging 18F-FDG with a modified gamma camera appears to be an effective alternative to imaging with a standard PET camera for assessing myocardial viability.

Lasting effects of one bout of two 15-second passive stretches on ankle dorsiflexion range of motion.

Although passive stretching is widely used, the parameters of stretching necessary to achieve a lasting length have not been determined. This study investigated the lasting effects of one bout of two 15-second passive stretches on ankle dorsiflexion range of motion. Conducting this study was important because, while it focused on a sufficient minimum duration, it considered lengthening mechanisms thought to contribute to a lasting length. Nineteen healthy volunteers with symmetrical limitations of ankle dorsiflexion participated. Stretching was done in unilateral standing with the subject's heel suspended over the edge of a platform. Four 5-second active dorsiflexion contractions were used as a preconditioning intended to stabilize the effects of mechanisms providing temporary length gains and were found to be effective. Measurements of passive dorsiflexion range of motion were taken over 24 hours. This study found no statistically significant length gains using a single bout of two 15-second stretches. These data do not provide evidence of lasting lengthening at this duration. Further research to determine a minimum one-bout lasting length duration is encouraged.

Pesticide residues in eggs and chicks from laying hens fed low levels of several chlorinated hydrocarbon pesticides.

Eighty-four Single-Comb While Leghorn laying hens housed individually in laying cages were fed rations containing less than 0.1 p.p.m. of dieldrin, DDT, heptachlor and mirex individually or in combination for 7 days and in combination for 15 weeks. DDT residues in egg yolk reached 0.043 p.p.m. by 7 days when fed in combination with the other pesticides. None of the residues were above FDA action level at 7 days and all had declined to below trace levels by 8 weeks after termination of pesticide feeding. Residues in eggs from hens fed all four pesticides for 15 weeks increased steadily for the first few weeks and then reached a plateau or increased only slightly until pesticide feeding was terminated. By the end of the 5th week of pesticide feeding all pesticides except DDT had exceeded FDA action levels for pesticides in eggs. DDT residues reached a level of 0.139 p.p.m. by 8 weeks and did not increase thereafter. Traces of the pesticides were still present 24 weeks after termination of pesticide feeding. The pesticides tested did not affect fertility of hatchability of eggs collected during the 14th and 15th weeks of pesticide feeding. Total carcass fat of chicks hatched from these eggs had 0.024 p.p.m. dieldrin, .049 p.p.m. DDT, .001 p.p.m. heptachlor epoxide and, 0.47 mirex at 1 day of age.

High dose chemotherapy with bone marrow or peripheral stem cell rescue is an effective treatment option for patients with relapsed or progressive Ewing's sarcoma family of tumours.

BACKGROUND: The outcome for patients with recurrent or progressive Ewing's sarcoma family of tumours (ESFT) is poor. High dose therapy (HDT) has been used for a number of years in an attempt to improve survival; however, evidence for the efficacy of this treatment remains limited. PATIENTS AND METHODS: Between 1992 and 2004, 33 patients with recurrent or progressive ESFT were treated with HDT with bone marrow (n=2), peripheral blood stem cell (n=30), or bone marrow and peripheral blood stem cell support (n=1), at a single institution. HDT was with busulphan and melphalan in 22 patients; melphalan and etoposide in seven patients, three with total body irradiation (TBI); melphalan in three patients (2 with TBI), and busulphan and cyclophosphamide in one patient. RESULTS: The 2 and 5 year event free survival was 42.5% (95% CI, 26-59%) and 38.2% (95% CI, 21-55%) respectively. There was one treatment related death from colitis, and grade 4 infection was observed in two patients. CONCLUSIONS: Long-term survival can be attained in patients with recurrent or refractory ESFT treated with HDT. However, this treatment is associated with severe toxicity. A need remains for prospective randomised clinical trials of HDT in this group of patients.

Renal assessment: back to basics.

Nurses caring for patients over time may observe subtle changes when performing physical assessments. Relating the observed changes to the function of various body systems allows prompt interventions. Nurses need to correlate their knowledge of physiology to assessment findings. This article will assist nurses in relating physical assessment findings to renal system physiology.

Depression in grandparents raising grandchildren: results of a national longitudinal study.

OBJECTIVES: To assess the effect of undertaking custodial care of a grandchild on grandparents' depression levels and to determine what characteristics are associated with higher depression levels among caregiving grandparents. DESIGN: A longitudinal national probability panel study: the National Survey of Families and Households. The first wave of data (n= 13 008) was collected in 1987 and 1988, and the second wave of data (n=10008) was collected from 1992 through 1994. SETTING: The survey was conducted in respondents' households in the coterminous United States. PARTICIPANTS: The subsample for this study was composed of 3111 respondents who reported being grandparents during the 1992-1994 interviews and for whom complete depression information was available. Of these grandparents, 158 were the primary caregivers for their grandchildren in the 1990s. MAIN OUTCOME MEASURES: Depression was measured using a modified version of the Center for Epidemiological Studies Depression Scale. RESULTS: Those who provide primary care for a grandchild are almost twice as likely to have levels of depressive symptoms above the traditional Center for Epidemiological Studies Depression Scale cut point of 16 (25.1% vs 14.5%). Even when controlling for baseline depression and demographic variables known to affect depressive symptoms, undertaking the care of a grandchild was associated significantly with higher depression levels in a multivariate prospective analysis (P<.01). Among caregiving grandparents, those who recently assumed caregiving responsibilities (P<.05) and women (P<.10) were more depressed and older respondents (P<.10) and those in good health (P<.001) were less depressed. CONCLUSIONS: Undertaking the primary care of a grandchild is associated with an increase in levels of depression. Particularly in light of the recent dramatic increase in the prevalence of grandparent caregiving in the United States, physicians need to explore familial role changes with midlife and older patients who have symptoms of depression. Special attention should be paid to the most at-risk subsets of grandparent caregivers: those who are new caregivers, those in poor health, those who are younger, and women.

Germinal center reaction.

The germinal center reaction is one critical outcome of helper T-cell-dependent antigen-specific B-cell responses. Germinal center reactions are the culmination of an orchestrated series of intercellular information exchanges discussed here as the serial synapsis model of adaptive immunity. The main purpose of the germinal center reaction is the development of B-cell memory through iterative cycles of somatic antigen receptor diversification and the selection of B cells with receptors of best fit. Recent studies provide insight into the regulation of these complex processes in vivo with new information on the cellular organization of the memory B-cell compartment.

Development and maintenance of a B220- memory B cell compartment.

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.