PTEN Contributes to Profound PI3K/Akt Signaling Pathway Deregulation in Dystrophin-Deficient Dog Muscle

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3β (GSK3β), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38δ and p38γ kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3β, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3β and PTEN activities. PTEN/GSK3β-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3β and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects 1 newborn boy in 3500. This recessive disease is caused by mutations in the dystrophin gene, resulting in total lack of the protein,^1,2,3 and is characterized by severe degeneration of muscle fibers, progressive paralysis, and death. Dystrophin is located under the sarcolemma of muscle fibers, and is associated with a complex comprising several integral, peripheral membrane and cytoplasmic proteins: the dystrophin-glycoprotein complex (DGC).^4,5,6,7 By providing a strong physical link between the cytoskeleton network and the extracellular matrix, the DGC ensures the integrity of skeletal muscle fibers. In the absence of dystrophin, the complex is destabilized and this integrity is lost.^5,8 However, the impaired structural role of the DGC alone may not be sufficient to account for the massive degenerative process observed in DMD muscles. Numerous observations suggest that signaling pathway alterations may also participate in DMD pathogenesis.Dystrophin and various DGC proteins have been demonstrated to interact with a number of signaling proteins, including growth factor receptor-bound protein 2,⁹ neuronal nitric oxide synthase,¹⁰ calmodulin,¹¹ focal adhesion kinase,¹² and caveolin-3.^13,14,15 Moreover, studies of the X chromosome-linked muscular dystrophy (mdx) mouse¹⁶ revealed modulations in mitogen-activated protein kinase (MAPK) signaling cascades, as dystrophic animals exhibited increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2)^17,18 and c-jun N-terminal kinases 1 and 2 (JNK1/2),^19,20,21 and decreased phosphorylation of p38.¹⁸ Also, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been shown to be affected in the mdx mouse, with increased synthesis and phosphorylation of Akt.^22,23In addition to the limited information related to the origin of signal perturbations in dystrophic muscle, almost no information is available regarding signaling pathways in clinically relevant animal models or human tissue samples.²³ It is noteworthy that the mdx mouse model of DMD is characterized by successive degeneration/regeneration processes, but does not exhibit the progressive muscle wasting and accumulation of connective tissue observed during the development of the human disease.^24,25,26 The Golden Retriever muscular dystrophy (GRMD) dog, characterized by rapidly progressive clinical dysfunction, severe muscle weakness, and abundant fiber necrosis, displays a disease progression that is far more similar to human DMD.^27,28In this study, we used antibody arrays to assess the global phosphorylation status of key proteins of the PI3K/Akt and MAPK signaling pathways in skeletal muscles of 4-month-old healthy and GRMD dogs. Our data indicated that Akt1, glycogen synthase kinase-3β (GSK3β) and p70S6K, as well as ERK1/2 and p38δ and γ kinases all displayed a decreased phosphorylation level in GRMD muscle. Western immunoblot, immunohistochemistry analysis, and enzymatic assays allowed us to confirm these results and demonstrated that they were associated with a reduction in Akt activity and with enhanced GSK3β expression and activity. Analysis of key enzymes involved in Akt regulation revealed that phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) was present at a much higher level and was more active in GRMD muscle. Moreover, immunohistochemistry analysis showed that all of the GSK3β-positive fibers observed in GRMD muscle sections exhibited a strong cytosolic labeling of PTEN, suggesting that the accumulation of the phosphatase could play a central role in PI3K/Akt signaling pathway deregulation. The observation of PTEN/GSK3β-positive fibers in muscle sections from 3- and 36-month-old GRMD dogs further demonstrated that both the early and late stages of the disease share deregulation of the pathway. Collectively, our findings highly suggest that alterations in PTEN exist in GRMD muscle, which leads to long-term and deep modulation of the PI3K/Akt signaling pathway.     

DNAX accessory molecule‐1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vγ9Vδ2 T cells

Human Vγ9Vδ2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway‐derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vγ9Vδ2 T‐cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in γδ T‐cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule‐1 (DNAM‐1) and CD96 were expressed by Vγ9Vδ2 T cells. The ligands Nectin‐like‐5 specific of both DNAM‐1 and CD96, and also Nectin‐2, an additional ligand of DNAM‐1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb‐mediated masking experiments that cytotoxicity against HCC cells as well as IFN‐γ production in γδ T cells were dependent on DNAM‐1. Our experiments indicated that Nectin‐like‐5 but not Nectin‐2 was involved in DNAM‐1‐dependent γδ T‐cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb‐mediated blockade that DNAM‐1 and NKG2D could cooperate in the cell lysis of HCC.     

Comparison of the 1996 and 2001 census data for Aboriginal and non-Aboriginal workers in health care occupations

To meet the unique health needs of Aboriginal peoples (First Nations, Inuit and Métis), it is important to increase and encourage Aboriginal representation in health care. One Federal initiative, the Aboriginal Health Human Resource Initiative (AHHRI) at Health Canada, focuses on: (1) increasing the number of Aboriginal people working in health careers; (2) adapting health care educational curricula to support the development of cultural competencies; and (3) improving the retention of health care workers in Aboriginal communities. A health care system that focuses on understanding the unique challenges, concerns, and needs of Aboriginal people can better respond to this specific population, which suffers disproportionately from ill health in comparison to their non-Aboriginal counterparts. This report examines the supply of Aboriginal health care providers in Canada, based on geographic region, area of residence, Aboriginal identity, and occupation. Findings are drawn from the 1996 and 2001 censuses from Statistics Canada. Quantitative results provide a greater understanding of labour force characteristics of First Nation, Inuit, Métis, and non-Aboriginal health providers.     

Cost-effectiveness of pneumococcal polysaccharide vaccination in adults: A systematic review of conclusions and assumptions

Streptococcus pneumoniae infections in adults are associated with substantial morbidity, mortality, and costs. A literature review was conducted to identify strengths and limitations of the cost-effectiveness of pneumococcal polysaccharide vaccine studies. A comparative analysis of the impact of model parameters on cost-effectiveness ratios was complemented by systematic assessment of the studies. We identified 11 economic evaluations of pneumococcal polysaccharide vaccine (PPV-23) in adults. In general, all 11 studies found that vaccination with PPV-23 is a cost-effective, and in some cases a cost-saving strategy for the prevention of invasive pneumococcal disease (IPD). The systematic assessment indicated that the results of the cost-effectiveness studies of PPV-23 are influenced by the values applied to vaccine efficacy, IPD incidence and case-fatality.     

Dihydropyrimidine dehydrogenase activity and the IVS14+1G>A mutation in patients developing 5FU-related toxicity

AIMS: To examine retrospectively the relationship between DPD phenotype/genotype and the intensity of 5FU toxicity. METHODS: One hundred and thirty-one case-reports (81 women, 50 men) with 5FU-related toxicity were analyzed. RESULTS: The lower the DPD activity (10-504 pmol min(-1) mg(-1)), the higher the toxicity grade was scored (P < 0.01). Toxicity-related deaths occurred in nine patients (eight women) who significantly expressed lower DPD activity than other patients. Two of the deceased patients had normal DPD activity. The IVS14+1G>A mutation, analyzed in 93 patients, was detected in two patients (nonlethal toxicity). CONCLUSIONS: The IVS14+1G>A mutation may not help prevent toxicity and patients with normal DPD activity may develop life-threatening 5FU toxicity.     

Unmet needs and health care utilization in young adults with cerebral palsy

PURPOSE: To gain insight into the unmet needs and utilization of health care of young adults with cerebral palsy (CP) and to explore relations between unmet needs, health care utilization and subject characteristics. METHOD: A cross-sectional study was performed in 29 young adults with CP without severe learning disabilities (IQ > 70). Subject characteristics such as age, gender, limb distribution, level of gross motor functioning, level of education and perceived participation and autonomy were measured. Outcome measures were the Southampton Needs Assessment Questionnaire, Impact on Participation and Autonomy and a questionnaire on health care utilization. RESULTS: Young adults with CP reported unmet needs mostly on information (79%), mobility (66%) and health care (66%). About half of the participants visited a rehabilitation physician (52%) or a physical therapist (55%) in the past year. Participants with lower levels of gross motor functioning were found to have more unmet needs and visited various health care professionals more often than young adults with higher levels of gross motor functioning. However, participants with higher levels of gross motor functioning still reported several unmet needs. CONCLUSIONS: Although young adults with CP frequently receive treatment from health care professionals, they indicate unmet needs with respect to several areas such as information on diagnosis, functional mobility and formal health care. In the treatment of young adults with CP, attention should be paid to these aspects.     

WGA allows the molecular characterization of a novel large CFTR rearrangement in a black South African cystic fibrosis patient

By performing extensive scanning of whole coding and flanking sequences of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, we had previously identified the CF-causing mutations in black South African patients of different ethnic groups suspected with the disease. Of ten samples analyzed, there were six remaining that had either one (n = 2) or two (n = 4) unidentified CFTR alleles that have now been tested for large rearrangements using a semiquantitative fluorescent PCR assay. A novel deletion encompassing CFTR exon 2 was detected in one patient who was heterozygous for the mutation 3120+1G>A. The Caucasian deletion involving the same exon [c.54-5811_c.164+2186del8108ins182] was ruled out. The DNA had been stored for more than 12 years and only minute quantities remained. We thus used a whole-genome amplification method based on multiple displacement amplification to generate sufficient amounts of DNA to characterize the intronic breakpoints and identify the deletion at the genomic level. Mapping and sequencing the breakpoint junctions revealed a novel large deletion [c.54-1161_c.164+1603del2875]. We have designed a simple test to specifically detect the presence or absence of this large rearrangement. This study reports the first large CFTR rearrangement in a black South African CF patient, further defining the molecular spectrum of CF that will be useful for improving genetic testing and counseling in this region.     

Nurses on the move: diversity and the work environment

Over 191 million people make up the international migrant population of today. Their numbers have doubled since 1970, with women now accounting for almost half. The migrant population has been transformed and is changing the very nature of society in both source and destination countries. Greater differences in culture, language, work relationships, and coping mechanisms - in short, greater diversity in society and the workplace - offer many opportunities for excellence in transcultural nursing but also provides fertile ground for discrimination, victimisation, harassment and isolation. This article explores and describes current nurse migration flows, the impact migration has on nurses and the value of positive practice environment for the full integration of international nurses.