Long-Term Outcomes in the Management of Painful Diabetic Neuropathy.

BACKGROUND: Painful diabetic neuropathy (PDN) is a frequent complication of diabetes mellitus. Current treatment recommendations are based on short-term trials, generally of ≤3 months' duration. Limited data are available on the long-term outcomes of this chronic disease. The objective of this study was to determine the long-term clinical effectiveness of the management of chronic PDN at tertiary pain centres. METHODS: From a prospective observational cohort study of patients with chronic neuropathic non-cancer pain recruited from seven Canadian tertiary pain centres, 60 patients diagnosed with PDN were identified for analysis. Data were collected according to Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials guidelines including the Brief Pain Inventory. RESULTS: At 12-month follow-up, 37.2% (95% confidence interval [CI], 23.0-53.3) of 43 patients with complete data achieved pain reduction of ≥30%, 51.2% (95% CI, 35.5-66.7) achieved functional improvement with a reduction of ≥1 on the Pain Interference Scale (0-10, Brief Pain Inventory) and 30.2% (95% CI, 17.2-46.1) had achieved both these measures. Symptom management included at least two medication classes in 55.3% and three medication classes in 25.5% (opioids, antidepressants, anticonvulsants). CONCLUSIONS: Almost one-third of patients being managed for PDN in a tertiary care setting achieve meaningful improvements in pain and function in the long term. Polypharmacy including analgesic antidepressants and anticonvulsants were the mainstays of effective symptom management.

The ethics of Canadian entry-to-practice pain competencies: how are we doing?

BACKGROUND: Although unrelieved pain continues to represent a significant problem, prelicensure educational programs tend to include little content related to pain. Standards for professional competence strongly influence curricula and have the potential to ensure that health science students have the knowledge and skill to manage pain in a way that also allows them to meet professional ethical standards. OBJECTIVES: To perform a systematic, comprehensive examination to determine the entry-to-practice competencies related to pain required for Canadian health science and veterinary students, and to examine how the presence and absence of pain competencies relate to key competencies of an ethical nature. METHODS: Entry-to-practice competency requirements related to pain knowledge, skill and judgment were surveyed from national, provincial and territorial documents for dentistry, medicine, nursing, pharmacy, occupational therapy, physiotherapy, psychology and veterinary medicine. RESULTS: Dentistry included two and nursing included nine specific pain competencies. No references to competencies related to pain were found in the remaining health science documents. In contrast, the national competency requirements for veterinary medicine, surveyed as a comparison, included nine pain competencies. All documents included competencies pertaining to ethics. CONCLUSIONS: The lack of competencies related to pain has implications for advancing skillful and ethical practice. The lack of attention to pain competencies limits the capacity of health care professionals to alleviate suffering, foster autonomy and use resources justly. Influencing professional bodies to increase the number of required entry-to-practice pain competencies may ultimately have the greatest impact on education and practice.

Proliferative lifespan is conserved after nuclear transfer.

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.

Treatment of chronic pain by long-acting opioids and the effects on sleep.

Chronic pain affects a substantial part of the population, and conveys a huge economic cost to society. Owing to its prevalence and adverse impact, it is of particular interest to clinicians, patients, and the pharmaceutical industry. Conversely, the effects of pain on sleep, sleep on pain, and opioid analgesics on sleep represent a large gap in our understanding, even though pain and sleep are closely linked, inter-related conditions. Chronic pain is often treated by opioid analgesics, which are often thought to promote restful sleep. Indeed it may be assumed that by relieving pain, sleep quality will improve concomitantly. In fact, the reality is much more complicated. The effects of opioids vary according to their formulation and duration of action, and have diverse effects on sleep processes. Despite the prevalence of this problem, there is a surprising paucity of data on the effects of opioids on sleep. This review attempts to summarize the links between pain and sleep, and to look at the studies with opioid analgesics, particularly those with extended-release formulations, that have investigated the effects of opioid analgesics on sleep.

Genetic modification of sheep by nuclear transfer with gene-targeted somatic cells.

For many years the lack of germline competent embryonic stem cell lines in livestock meant that the targeted modification of endogenous genes was not possible in these species. The demonstration that livestock could be cloned by nuclear transfer from cultured somatic cells has now provided an alternative route to accomplish gene targeting. This chapter describes protocols for culturing primary sheep fibroblasts, introducing and selecting targeted modifications into them and then using these modified cells in nuclear transfer experiments.

Long-Term Outcome of the Management of Chronic Neuropathic Pain: A Prospective Observational Study.

This prospective observational cohort study addressed the long-term clinical effectiveness of the management of chronic neuropathic noncancer pain at 7 Canadian tertiary pain centers. Patients were treated according to standard guidelines and were followed at 3, 6, 12, 18, and 24 months. Standard outcome measures for pain, mood, quality of life, and overall treatment satisfaction were administered, with the primary outcome measure designated as the composite of 30% reduction in average pain intensity and 1-point decrease in the mean Interference Scale Score (0-10) of the Brief Pain Inventory at 12 months relative to baseline. Of 789 patients recruited, mean age was 53.5 ± 14.2 years (55% female) and mean duration of pain was 4.88 ± 5.82 years. Mean average pain intensity (0-10) at baseline was 6.1 ± 1.9. All standard outcome measures showed statistically significant improvement at 12 months relative to baseline (P < .001). However, only 23.7% attained clinically significant improvement in pain and function at 12 months as the primary outcome measure. Univariable analyses showed poorer outcomes at 12-month follow-up with longer duration of pain (P = .002), greater cigarette use (P = .01), more disability compensation (P = .001), and higher opioid doses at baseline and at 12 months (P < .02). Our present treatment modalities provide significant long-term benefit in only about a quarter of patients with neuropathic pain managed at tertiary care pain clinics. Opioid therapy may not be beneficial for the long term. Perspective: Evidence-based treatment of chronic neuropathic pain provides long-term benefit in only about one-quarter of patients seen in tertiary care centers. Opioid therapy may not be beneficial.

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector.

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.

Development of novel selective cell ablation in the mammary gland and brain to study cell-cell interactions and chemoprevention.

We have generated transgenic mice which express the gene encoding Escherichia coli nitroreductase (NTR) specifically in the luminal epithelial cells of the mammary gland and the glial cells of the brain. The enzyme activates an antitumour drug CB 1954, to produce a cross-linking agent that kills all cells expressing the enzyme. We have shown that administration of the antitumour drug CB 1954 rapidly and selectively kills these cells. Original experiments demonstrated the ability to ablate the luminal cells in the mammary gland with no apparent bystander effect. Subsequently, astrocytes expressing nitroreductase under the targeting of the GFAP promoter were selectively ablated following administration of the prodrug CB 1954 produces a degeneration of granular neurones due to changes in glutamate levels. Recent experiments demonstrated inhibition of myc-dependent mammary tumours using the same enzyme (nitroreductase)-prodrug (CB 1954), combination. Owing to the ease of control of NTR-mediated cell ablation, we anticipate that this system will supersede herpes simplex virus type 1 thymidine kinase. There are widespread potential applications for this approach in the dissection of complex cellular interactions during development and in the adult organism. The present transgenic models also have important applications for the study in vivo of novel prodrugs that can be selected for variable degrees of bystander effects. Such studies will have particular significance for those groups advocating the use of NTR as an appropriate enzyme for gene-directed enzyme prodrug therapy by providing models of a wide range of human disease for mechanistic and therapeutic experimentation. The results clearly demonstrate that the model has potential to study chemoprevention and fundamental questions on cell-cell interactions in cell biology.

Distal transgene insertion affects CpG island maintenance during differentiation.

About half of all genes have a CpG island surrounding the promoter and transcription start site. Most promoter CpG islands are normally unmethylated in all tissues, irrespective of the expression level of the associated gene. Establishment of the appropriate patterns of DNA methylation in the genome is essential for normal development and patterns of gene expression. Aberrant methylation of CpG islands and silencing of the associated genes is frequently observed in cancer. One gene with a 5'-CpG island is cytoplasmic beta-actin, which is an abundantly expressed protein and a major component of microfilaments. Inserting a betageo cassette into the 3'-untranslated region of beta-actin gene led to widespread but not ubiquitous lacZ expression in mice heterozygous for the modified beta-actin allele. Surprisingly, embryos homozygous for this insertion died at mid-gestation. The modified beta-actin allele was expressed in undifferentiated embryonic stem cells but was turned off as these cells differentiate in vitro and in vivo. We demonstrate that the insertion affects the maintenance of the methylation status of the CpG island of the modified beta-actin allele in differentiated but not in undifferentiated embryonic cells. These data suggest that there is a two-step process to defining a CpG island, requiring both embryonic establishment and a signal that maintains the CpG island in differentiated cells. Furthermore, they indicate that features built into the CpG island are not sufficient to direct CpG island maintenance during differentiation.

Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos; they can be cultured indefinitely and differentiated into many cell types in vitro. These cells therefore have the ability to provide insights into human disease and provide a potential unlimited supply of cells for cell-based therapy. Little is known about the factors that are important for maintaining undifferentiated hESCs in vitro, however. As a tool to investigate these factors, transfected hES clonal cell lines were generated; these lines are able to express the enhanced green fluorescent protein (EGFP) reporter gene under control of the OCT4 promoter. OCT4 is an important marker of the undifferentiated state and a central regulator of pluripotency in ES cells. These OCT4-EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells. Expression of OCT4-EGFP is colocalized with endogenous OCT4, as well as the hESC surface antigens SSEA4 and Tra-1-60. In addition, the expression is retained in culture for an extensive period of time. Differentiation of these cells toward the neural lineage and targeted knockdown of endogenous OCT4 expression by RNA interference downregulated the EGFP expression in these cell lines, and this correlates closely with the reduction of endogenous OCT4 expression. Therefore, these cell lines provide an easy and noninvasive method to monitor expression of OCT4 in hESCs, and they will be invaluable for studying not only OCT4 function in hESC self-renewal and differentiation but also the factors required for maintenance of undifferentiated hESCs in culture.

Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells.

Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development.

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.

Inhibition of myc-dependent breast tumor formation in transgenic mice.

One of the most promising approaches for cancer gene therapy is the use of the so-called suicide genes, which encode prodrug-activating enzymes and render transduced cells more sensitive to prodrugs. The enzyme nitroreductase (NTR) converts prodrug CB1954 into a cytotoxic DNA interstrand cross-linking agent. We have established transgenic mice in which the pro-oncogene c-myc and NTR were fused to the internal ribosome entry site and coexpressed in luminal cells of the mammary gland under the control of mouse whey acidic protein (WAP) promoter to evaluate NTR mediated ablation of mammary tumors. More than 78% of transgenic females developed in situ or infiltrating carcinomas after three to four pregnancies. By contrast, if the transgenic female mice were given the prodrug CB1954 during their third lactation, the incidence of tumors decreased to less than 40% (P < 0.05). The total number of carcinomas was even more striking with 117 carcinomas identified in 14 non-ablated transgenics compared with only five in 15 treated animals (p < 0.05, student t test). C-myc induced pleomorphic nuclei and mitotic figures were seen as a field change in over 70% of the untreated transgenics compared to 20% in the treated group. Our results suggest that the enzyme pro-drug system NTR-CB1954 efficiently inhibit myc-dependent tumor formation and malignant progression in the mammary gland.

Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts.

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression.

Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development.

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50-200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.