A locus on chromosome 9p predisposes to a specific disease manifestation, acute anterior uveitis, in ankylosing spondylitis, a genetically complex, multisystem, inflammatory disease

OBJECTIVE: Uveitis or intraocular inflammation is a major cause of visual loss. Acute anterior uveitis (AAU) affects approximately 40% of patients with ankylosing spondylitis (AS) but also affects patients with no evidence of spondylarthritis. We sought to determine whether a unique genetic region could be implicated in a specific manifestation-AAU-of a multisystem, inflammatory, genetically complex disease, AS. METHODS: Individuals from families multiplex for AAU were genotyped at 400 markers representing the ABI PRISM linkage map MD-10, and at the HLA-B, DRB1, DQA1, DQB1, and DPB1 alleles. Among the family members with AAU, 76 affected sibpairs were analyzed (6 without concomitant AS, 12 discordant for AS, and 58 concordant for AS). Two-point and multipoint nonparametric linkage analyses were performed, and 1-parameter allele-sharing model logarithm of odds (LOD) scores were determined. RESULTS: As previously reported for AS, linkage at the major histocompatibility complex region (chromosome 6p21) was evident, exhibiting the highest multipoint LOD score (4.96 at marker HLA-B). Strong linkage was seen at a region on chromosome 9p21-9p24, with a LOD score of 3.72 at marker D9S157. When compared with a companion cohort of AS families, the linkage at this region was found in association with AAU but not with AS. A third region on chromosome 1q23-1q31 was observed to have suggestive linkage (LOD 2.05 at marker D1S238), which overlaps with a region associated with AS. CONCLUSION: This is the first study in which a genetic region for AAU has been identified by genome-wide scan. Even though AS was highly prevalent in this cohort of families, a locus at chromosome 9p21-9p24 was identified that uniquely associates with AAU. Identifying the genetic perturbation at this region may advance our understanding of the mechanisms involved in tissue-specific pathology of complex inflammatory diseases.     

Computerized tumor multinucleation index (MuNI) is prognostic in p16+ oropharyngeal carcinoma

BACKGROUNDPatients with p16⁺ oropharyngeal squamous cell carcinoma (OPSCC) are potentially cured with definitive treatment. However, there are currently no reliable biomarkers of treatment failure for p16⁺ OPSCC. Pathologist-based visual assessment of tumor cell multinucleation (MN) has been shown to be independently prognostic of disease-free survival (DFS) in p16⁺ OPSCC. However, its quantification is time intensive, subjective, and at risk of interobserver variability.METHODSWe present a deep-learning–based metric, the multinucleation index (MuNI), for prognostication in p16⁺ OPSCC. This approach quantifies tumor MN from digitally scanned H&E-stained slides. Representative H&E-stained whole-slide images from 1094 patients with previously untreated p16⁺ OPSCC were acquired from 6 institutions for optimization and validation of the MuNI.RESULTSThe MuNI was prognostic for DFS, overall survival (OS), or distant metastasis–free survival (DMFS) in p16⁺ OPSCC, with HRs of 1.78 (95% CI: 1.37–2.30), 1.94 (1.44–2.60), and 1.88 (1.43–2.47), respectively, independent of age, smoking status, treatment type, or tumor and lymph node (T/N) categories in multivariable analyses. The MuNI was also prognostic for DFS, OS, and DMFS in patients with stage I and stage III OPSCC, separately.CONCLUSIONMuNI holds promise as a low-cost, tissue-nondestructive, H&E stain–based digital biomarker test for counseling, treatment, and surveillance of patients with p16⁺ OPSCC. These data support further confirmation of the MuNI in prospective trials.FUNDINGNational Cancer Institute (NCI), NIH; National Institute for Biomedical Imaging and Bioengineering, NIH; National Center for Research Resources, NIH; VA Merit Review Award from the US Department of VA Biomedical Laboratory Research and Development Service; US Department of Defense (DOD) Breast Cancer Research Program Breakthrough Level 1 Award; DOD Prostate Cancer Idea Development Award; DOD Lung Cancer Investigator-Initiated Translational Research Award; DOD Peer-Reviewed Cancer Research Program; Ohio Third Frontier Technology Validation Fund; Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering; Clinical and Translational Science Award (CTSA) program, Case Western Reserve University; NCI Cancer Center Support Grant, NIH; Career Development Award from the US Department of VA Clinical Sciences Research and Development Program; Dan L. Duncan Comprehensive Cancer Center Support Grant, NIH; and Computational Genomic Epidemiology of Cancer Program, Case Comprehensive Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH, the US Department of VA, the DOD, or the US Government.     

Chronic Neuron- and Age-Selective Down-Regulation of TNF Receptor Expression in Triple-Transgenic Alzheimer Disease Mice Leads to Significant Modulation of Amyloid- and Tau-Related Pathologies

Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimer''s disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid β–driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti–TNF-α inhibition accelerates disease, cautions against long-term use of anti–TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector–delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI–mediated exacerbation of amyloid β and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1–positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti–TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti–TNF-α therapeutics for AD.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Alzheimer''s disease (AD) pathophysiology is described by chronic and progressive neurodegeneration involving the genesis of extracellular amyloid β (Aβ) plaques, intraneuronal filamentous inclusions called neurofibrillary tangles (NFTs), synapse loss, inflammation, and neuronal cell death, ultimately leading to severe memory loss and cognitive impairment. Neuroinflammation is a highly enigmatic process contributing to disease pathogenesis in AD, where elevated levels of proinflammatory molecules have been associated with Aβ-induced inflammation, neurotoxicity, and cognitive decline.^1–4 In AD-afflicted brains, microglia intimately co-localize with Aβ plaques and serve as major sources of proinflammatory mediators, including cytokines and chemokines.⁵ The pleiotropic proinflammatory cytokine tumor necrosis factor α (TNF-α) is produced in excess concurrently with increased Aβ plaque deposition, an observation that suggests that TNF-α levels reflect the pathologic progression of AD.^6–8 Moreover, three TNF-α promoter polymorphisms have been associated with late-onset AD, and two of the three polymorphisms are linked to increased TNF-α production, further connecting this cytokine to the exacerbated chronic inflammatory disease status in AD.⁹ We and others have demonstrated that TNF-α expression is enhanced in AD mouse models where TNF-α is prepathologically up-regulated in 6-month-old triple-transgenic AD (3xTg-AD) mice,^10,11 which corresponds with an enhancement of F4/80-positive microglial cell numbers.¹² In addition, when neuron-specific TNF-α is chronically overexpressed in 3xTg-AD mice using adeno-associated virus (AAV) vectors, there is increased severity of inflammation, intracellular Aβ, and Tau pathology that leads to neuronal cell death portending that excessive and unopposed TNF-α signaling enhances AD-associated pathology and is detrimental to neuronal viability.¹³TNF-α signals through two cognate transmembrane receptors, TNF receptor type I (TNF-RI) and TNF-RII, which are differentially expressed and regulated. TNF-RI is expressed constitutively on most cell types, whereas TNF-RII expression is induced and is restricted to specific cell populations, including hematopoietic cells, microglia, neurons, and endothelial cells.^14,15 TNF-R engagement to its ligand mediates distinct cellular responses through the activation of several downstream signal transduction cascades involving the NFκB and JNK pathways. In the context of AD, several reports demonstrate differential roles and activation of TNF-RI and TNF-RII such that genetic deletion of TNF-RI, but not TNF-RII, results in reduced plaque deposition in the APP23 mouse model.¹⁶ Moreover, in human brain tissue, TNF-RI protein levels are increased, whereas TNF-RII levels are reduced in patients with AD relative to nondemented control brain.¹⁷ Taken together, these data imply an overall negative role for excessive TNF signaling on AD pathophysiology but, perhaps more importantly, illustrate the complexity of this signaling pathway.Despite a large body of literature indicating detrimental roles for TNF-α, neuroprotective effects have also been reported. Early experiments revealed that TNF-α is protective in cultured neurons during glucose deprivation–induced injury and excitotoxicity by preserving Ca²⁺ homeostasis.¹⁸ Barger et al¹⁹ further demonstrated in dissociated neuronal cultures that pretreatment with TNF-α and Aβ peptide spares cells from Aβ-induced neuronal death, iron toxicity, and intracellular Ca²⁺ accumulation via an NF-κB–dependent mechanism. Moreover, neurons are vulnerable to ischemic injury and oxidative stress in TNF-R null mice, indicating that TNF-α is protective.²⁰ Mice lacking TNF-R expression exhibited reduced manganese superoxide dismutase activity and lacked a robust microglial response to kainic acid.²⁰ Similarly, cultured neurons pretreated with TNF-α resulted in a significant increase in manganese superoxide dismutase activity and a reduction in superoxide accumulation.²¹ These data add to the complexity of the TNF signaling pathway and suggest that strategies to modulate TNF-α in the disease setting may require selective tuning and specificity to ensure that protective signaling outcomes are not compromised.Nonetheless, given the compelling data supporting the pathologic role of TNF-α in AD, the potential of using anti–TNF-α therapeutics has become a viable strategy for subverting the disease course. Preclinical data by McAlpine et al²² demonstrate that transiently inhibiting soluble TNF signaling in the 3xTg-AD mouse model using a dominant-negative inhibitor in conjunction with enhanced systemic inflammation prevents AD-associated amyloid pathology. Tobinick et al²³ reported in a short-term, prospective, open-label pilot study that semiweekly perispinal administration of etanercept, a receptor decoy biological agent antagonizing the actions of TNF-α, in 15 patients with mild to severe AD led to significant and rapid cognitive improvements compared with untreated control patients as assessed by three separate tests measuring cognitive function.Although previous studies provide evidence suggesting that TNF-α inhibition in the short-term may lead to improved pathologic and functional outcomes, they lack data addressing the long-term consequences of blocking TNF-α in a global manner, where cell, stage, and receptor specificity were not examined. To this end, we recently demonstrated that long-term global inhibition of TNF-R signaling in 3xTg-AD mice where TNF-RI and TNF-RII were ablated in all cell types results in a robust increase in hallmark amyloid and NFT pathology. Furthermore, in the absence of TNF signaling, microglia seem nonresponsive to the developing amyloid pathology, which correlates with an impairment of microglial-mediated Aβ₄₂ phagocytosis activity in vitro.²⁴ These data suggest that caution should be taken with the use of broad long-term anti-TNF inhibitors and that a more selective strategy should be investigated.To add to our understanding of TNF signaling biology and the consequences of selectively modulating this pathway, we investigated the cell- and stage-specific role of TNF-R signaling in AD by using recombinant AAV (rAAV) vector–delivered siRNA technology to selectively knock down neuronal TNF-R signaling at stages preceding progressive pathology or in the presence of extant disease using the 3xTg-AD mouse model. We demonstrate that neuronal TNF-RI and TNF-RII exert differential actions where intact TNF-RII signaling results in suppressed Aβ plaque deposition and paired helical filament (PHF) formation in the context of progressive and established disease pathogenesis. In addition, we report a substantial reduction in Iba-1–positive microglia when rAAV2-delivered siTNF-RII or siTNF-RI+RII viral vectors are administered at 2 and 12 months of age. Taken together, these data demonstrate that selectively suppressing neuronal TNF-RI and/or TNF-RII leads to distinct and significant changes in AD pathogenesis, which is most likely a consequence of the divergent signaling pathways associated with these receptors. The present findings support further development and rigorous study of highly selective strategies designed to inhibit specific TNF-α–mediated signals and potentially disrupt the onset and/or progression of this debilitating disease.     

An international effort examining nursing student attitudes toward older people

Variations in culture and heritage can greatly impact an individual's beliefs, practices, and attitudes and may influence healthcare. The purpose of this research was to better understand the attitudes of student nurses toward older adults in the United States and Costa Rica. An exploratory quantitative research design was utilized for this study. Data were collected using the Kogan Old Persons scale from American and Costa Rican pre-licensure nursing students. Results revealed that total scores were very similar between students on the Kogan positive scale and the Kogan negative scale. Half of the individual items revealed significant differences between students of different cultures. Overall, nursing students reported positive attitudes toward older people with individual differences found between nationality and individual items. Understanding cultural variances and commonalities on student nurse attitudes toward older adults is important to the delivery of culturally diverse nursing education and culturally congruent care.     

Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.