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Olfactory dysfunction is an early and prevalent symptom of Alzheimer's disease (AD) and the olfactory bulb is a nexus of beta-amyloid plaque and tau neurofibrillary tangle (NFT) pathology during early AD progression. To mitigate the accumulation of misfolded proteins, an endoplasmic reticulum stress response called the unfolded protein response (UPR) occurs in the AD hippocampus. However, chronic UPR activation can lead to apoptosis and the upregulation of beta-amyloid and tau production. Therefore, UPR activation in the olfactory system could be one of the first changes in AD. In this study, we investigated whether two proteins that signal UPR activation are expressed in the olfactory system of AD cases with low or high amounts of aggregate pathology. We used immunohistochemistry to label two markers of UPR activation (p-PERK and p-eIF2α) concomitantly with neuronal markers (NeuN and PGP9.5) and pathology markers (beta-amyloid and tau) in the olfactory bulb, piriform cortex, entorhinal cortex and the CA1 region of the hippocampus in AD and normal cases. We show that UPR activation, as indicated by p-PERK and p-eIF2α expression, is significantly increased throughout the olfactory system in AD cases with low (Braak stage III-IV) and high-level (Braak stage V-VI) pathology. We further show that UPR activation occurs in the mitral cells and in the anterior olfactory nucleus of the olfactory bulb where tau and amyloid pathology is abundant. However, UPR activation is not present in neurons when they contain NFTs and only rarely occurs in neurons containing diffuse tau aggregates. We conclude that UPR activation is prevalent in all regions of the olfactory system and support previous findings suggesting that UPR activation likely precedes NFT formation. Our data indicate that chronic UPR activation in the olfactory system might contribute to the olfactory dysfunction that occurs early in the pathogenesis of AD.

Cerebral autosomal dominant arteriolopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is the most common form of hereditary small vessel disease (SVD) of the brain. Neuronal apoptosis has been demonstrated in the cortex of patients. Whether it is associated with an activation of the pro-apoptotic protein PKR pathway is unknown. Similarly, activation of autophagy in CADASIL has never been explored. Immunostaining of four CADASIL brains previously analyzed for cortical neuronal apoptosis and five control brains for PKR (phosphoPKR) and autophagy (ATG5, LC3II) activation markers. Significant nuclear pPKR staining was observed in CADASIL neurons comparatively to controls (p = 0.001). No difference was observed between patients and controls with autophagy markers. We demonstrated the activation of PKR pathway in CADASIL. This was not associated with a detectable modulation of autophagy. These results open a new field to explore in order to better understand the mechanisms underlying cortical neurons apoptosis.

In response to a variety of insults the unfolded protein response (UPR) is a major cell program quickly engaged to promote either cell survival or if stress levels cannot be relieved, apoptosis. UPR relies on three major pathways, named from the endoplasmic reticulum (ER) resident proteins IRE1α, PERK, and ATF6 that mediate response. Current tools to measure the activation of these ER stress response pathways in mammalian cells are cumbersome and not compatible with high-throughput imaging. In this study, we present IRE1α and PERK sensors with improved sensitivity, based on the canonical events of xbp1 splicing and ATF4 translation at ORF3. These sensors can be integrated into host cell genomes through lentiviral transduction, opening the way for use in a wide array of immortalized or primary mammalian cells. We demonstrate that high-throughput single-cell analysis offers unprecedented kinetic details compared with endpoint measurement of IRE1α and PERK activity. Finally, we point out the limitations of dye-based nuclear segmentation for live cell imaging applications, as we show that these dyes induce UPR and can strongly affect both the kinetic and dynamic responses of IRE1α and PERK pathways.

An important step in the initiation of the innate immune response to virus infection is the recognition of non-self, viral RNA, including double-stranded RNA (dsRNA), by cytoplasmic pattern recognition receptors (PRRs). For many positive-sense RNA viruses and DNA viruses, the production of viral dsRNA, and the interaction of viral dsRNA and PRRs are well characterized. However, for negative-sense RNA viruses, viral dsRNA was thought to be produced at low to undetectable levels and PRR recognition of viral dsRNA is still largely unclear. In the case of arenaviruses, the nucleocaspid protein (NP) has been identified to contain an exoribonuclease activity that preferentially degrades dsRNA in biochemical studies. Nevertheless, pathogenic New World (NW) arenavirus infections readily induce an interferon (IFN) response in a RIG-I dependent manner, and also activate the dsRNA-dependent Protein Kinase R (PKR). To better understand the innate immune response to pathogenic arenavirus infection, we used a newly identified dsRNA-specific antibody that efficiently detects viral dsRNA in negative-sense RNA virus infected cells. dsRNA was detected in NW arenavirus infected cells colocalizing with virus NP in immunofluorescence assay. Importantly, the dsRNA signals also colocalized with cytoplasmic PRRs, namely, PKR, RIG-I and MDA-5, as well as with the phosphorylated, activated form of PKR in infected cells. Our data clearly demonstrate the PRR recognition of dsRNA and their activation in NW arenavirus infected cells. These findings provide new insights into the interaction between NW arenaviruses and the host innate immune response.

Objective: Aggressive cutaneous squamous cell carcinoma (cSCC) patients are at increased risk of metastasis. Currently, there are no accepted criteria or biomarkers for reliably predicting individuals at risk for recurrence and metastasis. Our objective is to determine if pS6 and pERK can predict cSCC aggressiveness and to identify primary tumor characteristics that may predict parotid metastasis., Study Design: Retrospective case series., Settings: Tertiary care center., Subjects and Methods: An Institutional Review Board-approved retrospective review was performed for patients with facial cSCC, with and without metastasis to the parotids. Subjects for the study were identified through the Louisiana Tumor Registry, Veterans Medical Records, and LSU Health-Shreveport pathology database. Tumor specimens from patients with cSCC and cSCC with parotid metastasis were analyzed for pERK and pS6 expression through immunohistochemistry. To identify risk factors for tumor aggressiveness, multiple logistic regression analysis was used to evaluate patients with cSCC that was metastatic to the parotid and managed surgically., Results: cSCC with parotid metastasis specimens exhibited significantly higher average pS6 but not pERK positivity than those from cSCC without metastasis (P < .05). Primary lesion-positive margins (P < .01), size of the skin tumor (P < .01) and degree of tumor differentiation (P < .01) were significantly associated with parotid metastasis., Conclusion: Surgical history of cSCC, primary lesion-positive margins, degree of differentiation, and lesion size together with pS6 positivity appear to be predictors of cSCC aggressiveness and should prompt increased monitoring or elective parotidectomy., (© American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.)

Background: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways, leading to less protein production and activating genes that encode important proteins in folding processes, including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However, HIV infection's effect on the UPR has scarcely been investigated., Methods: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered., Results: In in vitro and in vivo analyses, our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile., Conclusions: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.

Zinc oxide nanoparticles (Nano-ZnO) are widely used in sunscreens, clothes, medicine and electronic devices. However, the potential risks of human exposure and the potential for adverse health impacts are not well understood. Previous studies have demonstrated that exposure to Nano-ZnO caused liver damage and hepatocyte apoptosis through oxidative stress, but the molecular mechanisms that are involved in Nano-ZnO-induced hepatotoxicity are still unclear. Endoplasmic reticulum (ER) is sensitive to oxidative stress, and also plays a crucial role in oxidative stress-induced damage. Previous studies showed that ER stress was involved in many chemical-induced liver injuries. We hypothesized that exposure to Nano-ZnO caused oxidative stress and ER stress that were involved in Nano-ZnO-induced liver injury. To test our hypothesis, mice were gavaged with 200 mg/kg or 400 mg/kg of Nano-ZnO once a day for a period of 90 days, and blood and liver tissues were obtained for study. Our results showed that exposure to Nano-ZnO caused liver injury that was reflected by focal hepatocellular necrosis, congestive dilation of central veins, and significantly increased alanine transaminase (ALT) and aspartate transaminase (AST) levels. Exposure to Nano-ZnO also caused depletion of glutathione (GSH) in the liver tissues. In addition, our electron microscope results showed that ER swelling and ribosomal degranulation were observed in the liver tissues from mice treated with Nano-ZnO. The mRNA expression levels of ER stress-associated genes (grp78, grp94, pdi-3, xbp-1) were also up-regulated in Nano-ZnO-treated mice. Nano-ZnO caused increased phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α). Finally, we found that exposure to Nano-ZnO caused increased ER stress-associated apoptotic protein levels, such as caspase-3, caspase-9, caspase-12, phosphorylation of JNK, and CHOP/GADD153, and up-regulation of pro-apoptotic genes (chop and bax). These results suggest that oxidative stress and ER stress-induced apoptosis are involved in Nano-ZnO-induced hepatotoxicity in mice., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background and Objective: Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1., Material and Methods: The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65)., Results: P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells., Conclusion: The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P. gingivalis through the NF-κB signaling pathway., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Amyloid β peptide (Aβ) immunization of Alzheimer's disease (AD) patients has been reported to induce amyloid plaque removal, but with little impact on cognitive decline. We have explored the consequences of Aβ immunotherapy on neurons in post mortem brain tissue. Eleven immunized (AN1792, Elan Pharmaceuticals) AD patients were compared to 28 non-immunized AD cases. Immunohistochemistry on sections of neocortex was performed for neuron-specific nuclear antigen (NeuN), neurofilament protein (NFP) and phosphorylated-(p)PKR (pro-apoptotic kinase detected in degenerating neurons). Quantification was performed for pPKR and status spongiosis (neuropil degeneration), NeuN-positive neurons/field, curvature of the neuronal processes and interneuronal distance. Data were corrected for age, gender, duration of dementia and APOE genotype and also assessed in relation to Aβ42 and tau pathology and key features of AD. In non-immunized patients, the degree of neuritic curvature correlated with spongiosis and pPKR, and overall the neurodegenerative markers correlated better with tau pathology than Aβ42 load. Following immunization, spongiosis increased, interneuronal distance increased, while the number of NeuN-positive neurons decreased, consistent with enhanced neuronal loss. However, neuritic curvature was reduced and pPKR was associated with Aβ removal in immunized patients. In AD, associations of spongiosis status, curvature ratio and pPKR load with microglial markers Iba1, CD68 and CD32 suggest a role for microglia in neurodegeneration. After immunization, correlations were detected between the number of NeuN-positive neurons and pPKR with Iba1, CD68 and CD64, suggesting that microglia are involved in the neuronal loss. Our findings suggest that in established AD this form of active Aβ immunization may predominantly accelerate loss of damaged degenerating neurons. This interpretation is consistent with in vivo imaging indicating an increased rate of cerebral atrophy in immunized AD patients., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic reticulum, commonly resulting in an accumulation of unfolded proteins in the ER lumen which is sensed by three ER-resident transmembrane proteins, PERK, ATF6, and IRE1. Their activation by such ER stress affects the unfolded protein response, which consists of a shutoff of protein translation and at the same time the switching-on of specific transcription factors that control genes which function to reduce the burden of unfolded proteins to the ER. Here, we describe two sets of methods for monitoring the occurrence of ER stress and UPR signaling in human cells by analyzing markers of activation of all three ER stress sensor proteins. The first set of methods is based on the qualitative and quantitative analysis of UPR-induced transcripts by qPCR. The second set of methods consists of Western blot-based analysis of UPR-induced proteins or protein modifications. Their combined analysis allows assessment of activation of all three ER stress-activated signaling pathways that in combination are characteristic for the UPR.

It has been known for some time that transplant recipients may have antibodies to endothelial cells which are not detected on lymphocytes. However, little progress has been made in the analysis of these endothelial antigens. In the present experiments we have attempted to characterize endothelial cell surface antigens to which antibodies were produced during graft rejection. We have used a panel of endothelial cells from umbilical cord veins and found that antibodies with a polymorphic pattern in the panel appeared to correlate with transplant failure of kidney allografts and with the development of transplant-related coronary artery disease (TCAD) in heart transplant recipients. Among 39 patients with kidney allografts, 21 were negative for antibodies to endothelial cells and did well and 18 were positive and had frequent transplant loss (p=0.001). In 18 patients with TCAD and 20 patients of a comparator group without TCAD, association of coronary disease with endothelial cell antibodies was observed (p<0.02). To characterize the endothelial antigens responsible for these serologic reactions we performed immunoprecipitation of reactive antibodies with the corresponding endothelial cell surface antigens, followed by protein identification of the target antigens. Nine proteins were identified in these experiments, 5 were non-polymorphic and appeared to represent autoantigens. Four of the isolated proteins appeared to be polymorphic. They were the Human Major Histocompatibility Complex class I chain-related gene A (MICA), already known to be associated with antibody production and graft failure, human keratin 1, a protein known to be polymorphic and expressed on the surface of endothelial cells, eukaryotic translation initiation factor (EIF) 2A and ErbB3-binding protein 1. The possible role of keratin 1 and the other antigens in allograft rejection requires further investigation., (Copyright © 2013. Published by Elsevier Inc.)

We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of protein kinase R (PKR). PKR's pro-apoptotic role through eukaryotic initiation factor 2 α (eIF2α) phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially, we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR's activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival nuclear factor-κB pathway. Thus, PKR has a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886's role in the PKR pathway during tumorigenesis.

Background and Objective: LL37, originally found in the innate immune system, is a robust antimicrobial peptide. LL37 exhibits multiple bio-functions in various cell types, such as migration, cytokine production, apoptosis, and angiogenesis besides its antimicrobial activity Periodontal ligament (PL) cells play a pivotal role in periodontal tissue regeneration. Based on these findings, we hypothesized that LL37 can regulate PL cell function to promote regeneration of periodontal tissue. To prove this hypothesis, we investigated the effect of LL37 on the potent angiogenic inducer vascular endothelial growth factor (VEGF) expression in cultures of human PL (HPL) cells because neovascularization is indispensable for the progress of tissue regeneration. Moreover, we investigated the signaling cascade associated with LL37-induced VEGF expression., Material and Method: HPL cells were treated with synthesized LL37 in the presence or absence of PD98059, a MEK-ERK inhibitor, or PDTC, an NF-κB inhibitor. VEGF expression levels were assessed by real-time polymerase chain reaction analysis and an enzyme-linked immunoassay. Phosphorylation levels of ERK1/2 or NF-κB p65 were determined by Western blotting., Results: LL37 upregulated VEGF-A expression at the mRNA and protein levels in HPL cells, while VEGF-B mRNA expression was not affected. Both ERK and NF-κB inhibitors clearly abrogated the increase in VEGF-A levels induced by LL37 in HPL cells. Importantly, LL37 increased phosphorylated levels of ERK1/2 and NF-κB p65 in HPL cells., Conclusion: LL37 induces VEGF-A production in HPL cells via ERK and NF-κB signaling cascades, which may result in angiogenesis, thereby contributing to periodontal regeneration., (© 2012 John Wiley & Sons A/S.)

The unfolded protein response (UPR) is a stress response activated upon disturbed homeostasis in the endoplasmic reticulum (ER). Previously, we reported that the activation of the UPR closely correlates with the presence of phosphorylated tau (p-tau) in Alzheimer's disease (AD). As well as increased presence of intracellular p-tau, AD brains are characterized by extracellular deposits of β amyloid (Aβ). Recent in vitro studies have shown that Aβ can induce ER stress and activation of the UPR. The aim of the present study is to investigate UPR activation in sporadic tauopathies like progressive supranuclear palsy (PSP) and Pick's disease (PiD), and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) which carry mutations in the gene encoding for tau (MAPT). The presence of phosphorylated pancreatic ER kinase (pPERK) and phosphorylated inositol requiring enzyme 1α (pIRE1), which are indicative of an activated UPR, was assessed by immunohistochemistry in cases neuropathologically defined as frontotemporal lobar degeneration with tau pathology (FTLD-tau). Increased presence of UPR activation markers pPERK and pIRE1 was observed in neurons and glia in FTLD-tau cases, in contrast to FTLD subtypes negative for tau pathology or in non-neurological controls. pPERK and pIRE1 were also prominently present in relatively young carriers of MAPT mutation. A strong association between the presence of UPR activation markers and p-tau was observed in the hippocampus of FTLD-tau cases. Double immunohistochemical staining on FTLD-tau cases revealed that UPR activation is predominantly observed in neurons that show diffuse staining of p-tau. These data demonstrate that UPR activation is intimately connected with the accumulation and aggregation of p-tau, and occurs independently from Aβ deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2 alpha (eIF2 alpha) kinase is activated in acute heme-deficient states, resulting in translational shut-off of global hepatic protein synthesis, including phenobarbital (PB)-mediated induction of CYP2B enzymes in rats. These findings revealed that heme regulates hepatic CYP2B synthesis at the translational level via HRI. As a proof of concept, we have now employed a genetic HRI-knockout (KO) mouse hepatocyte model. In HRI-KO hepatocytes, PB-mediated CYP2B protein induction is no longer regulated by hepatic heme availability and proceeds undeterred even after acute hepatic heme depletion. It is noteworthy that genetic ablation of HRI led to a small albeit significant elevation of basal hepatic endoplasmic reticulum (ER) stress as revealed by the activation of ER stress-inducible RNA-dependent protein kinase-like ER-integral (PERK) eIF2 alpha-kinase, and induction of hepatic protein ubiquitination and ER chaperones Grp78 and Grp94. Such ER stress was further augmented after PB-mediated hepatic protein induction. These findings suggest that HRI normally modulates the basal hepatic ER stress tone. Furthermore, because HRI exists in both human and rat liver in its heme-sensitive form and is inducible by cytochrome P450 inducers such as PB, these findings are clinically relevant to acute heme-deficient states, such as the acute hepatic porphyrias. Activation of this exquisitely sensitive heme sensor would normally protect cells by safeguarding cellular energy and nutrients during acute heme deficiency. However, similar HRI activation in genetically predisposed persons could lead to global translational arrest of physiologically relevant enzymes and proteins, resulting in the severe and often fatal clinical symptoms of the acute hepatic porphyrias.

Type 1 diabetes is caused by autoimmune destruction of pancreatic beta cells, possibly virus initiated. Virus infection induces alpha-interferon (IFN-alpha), leading to upregulation of genes encoding double-stranded (ds) RNA-dependent antiviral enzymes 2', 5'-oligoadenylate synthetase (2'5'AS) and PKR (p68). To investigate whether beta cell specificity could be due to antiviral differences between beta and alpha cells, we treated beta and alpha TC3 cell lines with IFN-alpha and/or poly(I:C) (a synthetic dsRNA). Results showed that, following IFN-alpha stimulation, increases in 2'5'AS levels and activities were significantly higher in beta than alpha cells (P < .001), whereas increases in PKR level and activity were comparable in the two cell types. Poly(I:C) stimulated 2'5'AS activity in beta but not alpha cells, and co-transfection IFN-alpha plus poly(I:C) induced apoptosis in beta but not alpha cells. These findings suggest that the elevated 2'5'AS response of pancreatic beta cells could render them particularly vulnerable to damage and/or apoptosis during virus infection.

Innate immunity is a constitutive component of the central nervous system (CNS) and relies strongly on resident myeloid cells, the microglia. However, evidence is emerging that the most abundant glial cell population of the CNS, the astrocyte, participates in the local innate immune response triggered by a variety of insults. Astrocytes display an array of receptors involved in innate immunity, including Toll-like receptors, nucleotide-binding oligomerization domains, double-stranded RNA-dependent protein kinase, scavenger receptors, mannose receptor and components of the complement system. Following activation, astrocytes are endowed with the ability to secrete soluble mediators, such as CXCL10, CCL2, interleukin-6 and BAFF, which have an impact on both innate and adaptive immune responses. The role of astrocytes in inflammation and tissue repair is elaborated by recent in vivo studies employing cell-type specific gene targeting.

Rhinovirus infections cause the majority of acute exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease, with increased pro-inflammatory cytokine production by infected bronchial epithelial cells contributing to disease pathogenesis. Theses diseases are a huge cause of morbidity worldwide, and contribute a major economic burden to healthcare costs. Current steroid based treatments are only partially efficient at controlling virus induced inflammation, which remains an unmet therapeutic goal. Although NF-kappaB has been implicated, the precise mechanisms of rhinovirus induction of pro-inflammatory gene expression in bronchial epithelial cells are unclear. We hypothesised that rhinovirus replication and generation of dsRNA was an important process of pro-inflammatory cytokine induction. Using pharmalogical (2-aminopurine and a new small molecule inhibitor) and genetic inhibition of the dsRNA binding kinase protein kinase R, striking inhibition of dsRNA (polyrIC) and rhinovirus induced CCL5, CXCL8 and IL-6 protein was observed. Using confocal microscopy, rhinovirus induced protein kinase R phosphorylation co-located with NF-kappaB p65 nuclear translocation. Focusing on CXCL8, both rhinovirus infection and dsRNA treatment required IkappaB kinase-beta for induction of CXCL8. Analysis of cis-acting sites in the CXCL8 promoter revealed that both rhinovirus infection and dsRNA treatment upregulated CXCL8 promoter activation via NF-kappaB and NF-IL6 binding sites. Together, the results demonstrate the importance of dsRNA in induction of pro-inflammatory cytokines by rhinoviruses, and suggest that protein kinase R is involved in NF-kappaB mediated gene transcription of pro-inflammatory cytokines via IkappaB kinase-beta. These molecules regulating rhinovirus induction of inflammation represent therapeutic targets.

The mammalian innate immune system provides a first line of defense against microbial pathogens and also serves to activate an antigen specific acquired immune program. Key components of innate immunity are the interferons (IFNs), a family of related cytokines with potent antimicrobial and immuno-modulatory activities. The IFNs exert their effects through the induction of numerous genes, one of which is the double-stranded RNA-dependent protein kinase (PKR), a pivotal antiviral protein found in most human cells. Following activation by double stranded (ds) RNAs produced during viral replication, PKR phosphorylates the alpha-subunit of eukaryotic translation initiation factor (eIF) 2, causing a severe inhibititon of cellular and viral protein synthesis. Phosphorylation of eIF2alpha and consequent inhibition of protein synthesis is a major cell growth checkpoint utilized by at least three other kinases, in addition to PKR, following exposure to such cellular stresses as amino acid deprivation and the presence of misfolded proteins in the endoplasmic reticulum. Indeed, it has been demonstrated that disruption of the eIF2alpha checkpoint can lead to the transformation of immortalized rodent and human cells, plausibly by increasing the protein synthesis rates of proto-oncogenes. Further, it has been shown that disregulation of the eIF2alpha checkpoint and consequent permissiveness to virus infection may be a common occurrence in tumorigenic mammalian cell lines. These findings have been exploited to develop potent oncolytic RNA viruses that can selectively replicate in and destroy a variety of neoplasias in vitro and in vivo. In this chapter, we describe some of the techniques commonly used in our laboratory to examine PKR activity and eIF2 regulation. Protocols for the generation and use of recombinant vesicular stomatitis virus variants are also described.

Aim: To study the amino acid substitutions in the carboxy (C)-terminal part of E2 protein and in the interferon (IFN) sensitivity determining region (ISDR) and their correlation with response to IFN and viral load in 85 hepatitis C virus (HCV)-1b-infected patients treated with IFN., Methods: The C-terminal part of E2 (codons 617-711) including PKR/eIF2alpha phosphorylation homology domain (PePHD) and ISDR was sequenced in 85 HCV-1b-infected patients treated by IFN monotherapy., Results: The amino acid substitutions in PePHD detected only in 4 of 85 patients were not correlated either with response to IFN or with viral load. The presence of substitutions in a N-terminal variable region (codons 617-641) in the C-terminal part of E2 was significantly correlated with both small viral load (33.9% vs 13.8%, P = 0.0394) and sustained response to IFN (25.0% vs 6.9%, P = 0.0429). Four or more substitutions in ISDR were significantly correlated with both small viral load (78.6% vs 16.2%, P < 0.0001) and sustained response to IFN (85.7% vs 2.9%, P < 0.0001). In multivariate analysis, ISDR in nonstructural (NS) 5A (OR = 0.39, P < 0.0001) and N-terminal variable region (OR = 0.51, P = 0.039) was selected as the independent predictors for small viral load, and ISDR (OR = 39.0, P < 0.0001) was selected as the only independent predictor for sustained response., Conclusion: The N-terminal variable region in the C-terminal part of E2 correlates with both response to IFN monotherapy and viral load and is one of the factors independently associated with a small viral load.

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58(IPK), the cellular inhibitor of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2alpha phosphorylation and unphosphorylated eIF2alpha is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2alpha phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2alpha kinases and phosphorylatable eIF2alpha. When eIF2alpha is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2alpha phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2alpha phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-gamma (IFN-gamma). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-alpha) concentration. Lysates of cells were subjected to Western blot analyses of IKK-alpha, IKK-beta, p65 (RelA) subunit of nuclear factor kappaB (NF-kappaB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-gamma and on AM without IFN-gamma. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-gamma activation, and this phenomenon became apparent only after 48 hr. IFN-gamma-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-alpha during 72 hr culturing. In contrast, there was no NO and TNF-alpha production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-gamma-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-alpha, IKK-beta, p65 (RelA) subunit of NF-kappaB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-gamma-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-gamma activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-kappaB and Akt-FKHR signalling pathways.

The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection.

The authors investigated the protein expression of double-stranded RNA-activated protein kinase (PKR), which was identified by using a previous cDNA microarray study, to discover PKR's correlations with several pathological parameters and to elucidate its role in neoplastic transformation and progression of lung adenocarcinomas. Immunohistochemistry for PKR was performed and a semiquantitative scoring method was calculated based on staining intensity and percentage of immunoreactive tumor cells (high vs low) for one bronchioloalveolar carcinoma (BAC), 16 adenocarcinomas consisting of BAC and invasive carcinoma (mixed) and 21 invasive adenocarcinomas without BAC (invasive). The BAC had high-grade expression and the mixed type tended to more frequently show high-grade expression than the invasive type (P = 0.028). There were no significant associations with age, tumor size, lymph node metastasis, lymphovascular invasion or the pathological stage. The Kaplan-Meier survival curves demonstrated that the patients with high-grade PKR expression had significantly shorter survival periods than those patients with low-grade PKR expression (P = 0.018). These results do not support the concept of PKR as a tumor suppressor in small-size peripheral adenocarcinomas of the lung.

The heme-regulated inhibitor of protein synthesis (HRI) regulates translation through the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF 2). While HRI is best known for its activation in response to heme-deficiency, we recently showed that the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD) of HRI activated and suppressed its activity, respectively. Here, we examined the effect of hemin, NO, and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/Delta HBD). Hemin stabilized the interaction of NT-HBD with HRI/Delta HBD, and NO and CO disrupted and stabilized this interaction, respectively. Mutant HRI (Delta H-HRI), lacking amino acids 116-158 from the NT-HBD, was less sensitive to heme-induced inhibition, and mutant NT-HBD lacking these residues did not bind to HRI/Delta HBD. HRI/Delta HBD and Delta H-HRI also activated more readily than HRI in response to heme-deficiency. Thus, HRI's activity is regulated through the modulation of the interaction between its NT-HBD and catalytic domain.

Interferon (IFN)-alpha has been used in the treatment of neuroendocrine (NE) tumors; however, the feasibility of IFN-alpha gene therapy has not been evaluated in NE tumor cells. In this study, human IFN-alpha2 (hIFN-alpha2) gene has been transferred into a NE tumor cell line BON. hIFN-alpha2-expressing BON cells were subcutaneously inoculated in nude mice. The results demonstrated that hIFN-alpha2 exerted significant antiproliferative effects on NE tumor cell lines (BON and LCC18) and other tumor cell lines (CA46 and SW480) as well as porcine aorta cell line. Furthermore, hIFN-alpha2 demonstrated its antineovascular activity in mice tumor and a direct antiangiogenic effect in chicken chorioallantoic membrane assay. hIFN-alpha2-expressing BON cells had a stable and long-term expression. Mice implanted with hIFN-alpha2-expressing BON cells showed a lower incidence, a delayed development and a significantly longer doubling time of the tumor compared to both wild-type (WT) and vector group. In addition, IFN-alpha significantly inhibited cell adhesion of WT BON cells. hIFN-alpha2-expressing BON tumors had a high level of hIFN-alpha2 protein. Finally, mice implanted with a mixture of WT and hIFN-alpha2-expressing BON cells (1:1) presented a delayed tumor development and had an even lower incidence of tumors than those implanted with hIFN-alpha2-expressing BON cells only. The doubling time of tumor was also longest in the mixture group. Our data suggest that hIFN-alpha2 gene therapy might be possible to be used as a new treatment for NE tumor patients. Further studies on the regulation of hIFN-alpha expression are needed, especially in combination with other cytokines, which could lead to a better understanding and improvements of hIFN-alpha gene therapy.

Breast cancer incidence increases with age but this relationship has not been fully explored with regard to expression of estrogen receptor (ER) and ER-inducible genes (PR, pS2, Bcl2, cathepsin D), or the age-dependence of oxidant stress markers that also affect ER-inducible gene expression. In this three-part study, we first correlated age at diagnosis with expression of breast cancer markers ER, PR, pS2, Bcl2, and cathepsin D, quantitated by enzyme immunoassays from a European collective of approximately 3000 cryobanked primary breast cancers and approximately 300 adjacent non-malignant breast tissues. Results were then compared with ER and PR data reported to the SEER registry for 83,541 US cancers diagnosed during 1992-1997. Lastly, a homogeneous subset of 70 ER-positive tumors preselected from the European collective was blindly analyzed for age-specific changes in the DNA-binding content of redox-sensitive transcriprtion factors, AP1 and Sp1, and the oxidant stress-activated protein kinase, phosphorylated(P)-Erk5. Increases in breast tumor ER from patients aged <30 to >80 years mirrored 10-fold lower increases in non-malignant breast tissue ER content up to age 60, rising faster thereafter and reaching a near 25-fold differential between malignant and non-malignant breast tissue by age 80. ER-inducible markers PR, pS2, Bcl2, and cathepsin D were overexpressed in tumors relative to non-malignant breast tissue but, unlike ER, did not increase with patient age. While SEER data demonstrated that the increase in US breast cancer incidence rates after age 50 is confined to ER-positive tumors in patients of all ethnic subsets, these patients also showed a striking increase in the proportion of higher-risk ER-positive/PR-negative breast cancers arising after age 50. Mechanistically essential for ER-inducible PR expression, Sp1 DNA-binding function (but not Sp1 content) was lost with age in ER-positive tumors; and this functional defect correlated with increased tumor content of the oxidant stress marker, P-Erk5. Altogether these findings support two hypotheses: (i) dysregulated ER expression underlies the age-specific increase in breast cancer incidence after age 50; and (ii) oxidative stress and loss of Sp1 DNA-binding may contribute to an increasing incidence in higher-risk ER-positive/PR-negative breast cancers with aging.

There is a large amount of evidence describing the expression, interaction, and mode of activation of the human interferon (IFN)-mediated double-stranded RNA-activated protein kinase (PKR) gene. Studies from Pkr-null mice have defined the kinase as a transducer of dsRNA signals that converge on transcription, translation, and apoptotic programs involved in the innate resistance to viral infection. In vitro studies also suggest that PKR may possess important cell growth regulatory and tumor suppressor properties. However, the study of Pkr-null mice has not fully elucidated the role that the kinase plays in these processes, in part because of apparent redundancies in PKR-dependent and PKR-independent regulatory pathways. To overcome such limitations and to begin to examine the role of PKR in a complex biologic system, we have generated transgenic mice overexpressing wild-type human (Hu) PKR. HuPKR was expressed and active in various tissues and associated with a small body phenotype. Spleen cells from transgenic mice were resistant to apoptosis when treated with the genotoxic agent actinomycin D and showed a decrease in proliferation in response to concanavalin A (ConA) compared with spleen cells from wild-type control mice. The initial characterization of this transgenic mouse line suggests it may be useful as a model for investigating biology and diseases relative to a number of scientific disciplines.

Double-stranded RNA (dsRNA) plays a role in the regulation of cell growth and apoptosis as well as in the cellular antiviral responses. However, it remains unknown if dsRNA-activated signaling systems are functional in the thyroid. Here we report the presence of the dsRNA-dependent protein kinase (PKR) in FRTL-5 rat thyroid cells. In poly(I)-poly(C) (pIC)-stimulated cells, activation of nuclear factor-kappa B (NF kappa B) binding was clearly induced. Incubation of FRTL-5 cells with pIC resulted in a marked increase in interferon regulatory factor-1 (IRF-1) mRNA and phosphorylated signal transducer and activator of transcription-1 (STAT1) levels. Addition of pIC to cells led to type I interferon (IFN) gene expression, especially IFN beta, which can induce STAT1 phosphorylation, suggesting that dsRNA indirectly induced STAT1 phosphorylation through expression of type I IFN. Thus, our results suggest that the dsRNA-activated signaling pathway may be involved in the regulation of IFN-inducible genes in the thyroid.

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-gamma mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-gamma inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-gamma on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells.

A method to quantify double-stranded RNA-dependent protein kinase (PKR) mRNA and protein from human cells is described. A competitive RT-PCR assay has been developed by synthesis of an internal standard control (ISC) species of RNA. A competitive immunoblot assay was used to quantify full-length PKR (FL-PKR) protein in a sample of total cellular proteins, using truncated PKR protein as an internal standard against which FL-PKR protein could be quantified. The method can be used for simultaneous analysis of transcriptional and postranscriptional regulation of PKR gene expression from very small clinical specimens such as liver biopsies, e.g., 2-3 mg (wet weight) and containing only 2x10(5) cells. To the best of our knowledge, this is the first report of a sensitive simultaneous assay system for this important immunoeffector molecule.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha), we used an antibody specific to phosphorylated eIF-2 alpha [eIF-2(alpha P)] to study the regional and cellular distribution of eIF-2(alpha P) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that approximately 1% of all eIF-2 alpha is phosphorylated in controls, eIF-2(alpha P) is not reduced by up to 30 minutes of ischemia, and eIF-2(alpha P) is increased approximately 20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(alpha P) to astrocytes in normal brains, a massive increase in eIF-2(alpha P) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(alpha P) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(alpha P) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(alpha P) inhibits translation initiation, make a compelling case that eIF-2(alpha P) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.