Alison H. Skalet

Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response

ABSTRACT Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cells metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.

Antibiotic Selection Pressure and Macrolide Resistance in Nasopharyngeal Streptococcus pneumoniae: A Cluster-Randomized Clinical Trial

Jeremy Keenan and colleagues report that during a cluster-randomized clinical trial in Ethiopia, nasopharyngeal pneumococcal resistance to macrolides was significantly higher in communities randomized to receive azithromycin compared with untreated control communities.

Telemedicine screening for retinopathy of prematurity in developing countries using digital retinal images: a feasibility project.

PURPOSE To determine the feasibility in a middle-level human development country of onsite training, image collection, Internet transfer, and remote grading of digital retinal images from babies screened for retinopathy of prematurity (ROP). METHODS Two experienced nurses in a neonatal nursery in Lima, Peru, were trained to take posterior pole (30 degrees ) digital retinal images. Nurses obtained posterior pole retinal images from babies undergoing routine ROP screening and selected images for uploading via Internet for remote evaluation by five masked ROP experts. Results of gradings were compared with same-day clinical diagnostic examinations by an experienced ophthalmologist. Success rates for image acquisition and transfer for grading by expert readers were calculated. RESULTS Serial image sets from 26 of the 28 babies enrolled in this study were obtained; two babies were too unstable for imaging. Fifty-six of 58 (96.6%) imaging sessions were successful in obtaining retinal images. Three hundred thirty of 336 (98.2%) images obtained were successfully uploaded to an interactive database. Remote graders judged 93.6% to 97.3% of image sets suitable for ROP grading. Preliminary results indicate sensitivities for detection of serious ROP from 45.5% to 95.2% among individual readers, with specificities of 61.7% to 96.2% when images were gradable. CONCLUSIONS A telemedicine approach for ROP screening using digital retinal images obtained by nonophthalmologists is feasible in rapidly developing countries that lack ROP-trained ophthalmologists. If remote grading of digital images is validated as an effective method for identifying referral-warranted ROP (RW-ROP), images obtained by nonphysicians may provide a means of identifying babies who require a diagnostic examination by an ophthalmologist.

Rapid B Cell Receptor-induced Unfolded Protein Response in Nonsecretory B Cells Correlates with Pro- Versus Antiapoptotic Cell Fate

The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop–/– B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli.

Accuracy and Reliability of Telemedicine for Diagnosis of Cytomegalovirus Retinitis

PURPOSE To determine the feasibility of remote diagnostic screening for cytomegalovirus (CMV) retinitis among HIV patients in northern Thailand. DESIGN Prospective, observational cross-sectional study. METHODS One hundred eighty-two eyes from 94 consecutive patients with HIV seen in 2008 and 2009 at a tertiary university-based medical center were photographed using a digital retinal camera. Individual and composite images were uploaded to a secure web site. Three expert graders accessed the electronic images and graded each image for signs of CMV retinitis. Results of remote expert grading were compared with on-site patient examination by local expert ophthalmologists. RESULTS On-site ophthalmologists diagnosed CMV retinitis in 89 (48.9%) of 182 eyes. Trained ophthalmic photographers obtained digital retinal images for all 182 eyes. As compared with the on-site examinations, the sensitivity for detecting CMV retinitis by remote readers using composite retinal images ranged from 89% to 91%. The specificity for detecting CMV retinitis by remote readers ranged from 85% to 88%. Intrarater reliability was high, with each grader achieving a κ value of 0.93. Interrater reliability among the 3 graders also was high, with a κ value of 0.86. CONCLUSIONS Remote diagnostic screening for CMV retinitis among HIV-positive patients may prove to be a valuable tool in countries where the burden of HIV exceeds the capacity of the local eye care providers to screen for ocular opportunistic infections.

Clinical features of newly diagnosed cytomegalovirus retinitis in northern Thailand.

PURPOSE To characterize the clinical manifestations of cytomegalovirus (CMV) retinitis in northern Thailand. DESIGN Prospective, observational, cross-sectional study. METHODS We recorded characteristics of 52 consecutive patients newly diagnosed with CMV retinitis at a tertiary university-based medical center in northern Thailand. Indirect ophthalmoscopy by experienced ophthalmologists was supplemented with fundus photography to determine the proportion of eyes with various clinical features of CMV retinitis. RESULTS Of the 52 patients with CMV retinitis, 55.8% were female. All were HIV-positive. The vast majority (90.4%) had started antiretroviral therapy. CMV retinitis was bilateral in 46.2% of patients. Bilateral visual acuity worse than 20/60 was observed in 23.1% of patients. Of 76 eyes with CMV retinitis, 61.8% had zone I disease and 21.6% had lesions involving the fovea. Lesions larger than 25% of the retinal area were observed in 57.5% of affected eyes. CMV retinitis lesions commonly had marked or severe border opacity (47.4% of eyes). Vitreous haze often was present (46.1% of eyes). Visual impairment was more common in eyes with larger retinitis lesions. Retinitis lesion size, used as a proxy for duration of disease, was associated with fulminant appearance (odds ratio, 1.24; 95% confidence interval, 1.01 to 1.51) and marked or severe border opacity (odds ratio, 1.36; 95% confidence interval, 1.11 to 1.67). Based on lesion size, retinitis preceded antiretroviral treatment in each patient. CONCLUSIONS Patients seeking treatment at a tertiary medical center in northern Thailand had advanced CMV retinitis, possibly because of delayed diagnosis. Earlier screening and treatment of CMV retinitis may limit progression of disease and may prevent visual impairment in this population.

Retinoblastoma and Neuroblastoma Predisposition and Surveillance

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. Approximately 40% of retinoblastomas are hereditary and due to germline mutations in the RB1 gene. Children with hereditary RB are also at risk for developing a midline intracranial tumor, most commonly pineoblastoma. We recommend intensive ocular screening for patients with germline RB1 mutations for retinoblastoma as well as neuroimaging for pineoblastoma surveillance. There is an approximately 20% risk of developing second primary cancers among individuals with hereditary RB, higher among those who received radiotherapy for their primary RB tumors. However, there is not yet a clear consensus on what, if any, screening protocol would be most appropriate and effective. Neuroblastoma (NB), an embryonal tumor of the sympathetic nervous system, accounts for 15% of pediatric cancer deaths. Prior studies suggest that about 2% of patients with NB have an underlying genetic predisposition that may have contributed to the development of NB. Germline mutations in ALK and PHOX2B account for most familial NB cases. However, other cancer predisposition syndromes, such as Li–Fraumeni syndrome, RASopathies, and others, may be associated with an increased risk for NB. No established protocols for NB surveillance currently exist. Here, we describe consensus recommendations on hereditary RB and NB from the AACR Childhood Cancer Predisposition Workshop. Clin Cancer Res; 23(13); e98–e106. ©2017 AACR. See all articles in the online-only CCR Pediatric Oncology Series.

Optical Coherence Tomography Angiography Characteristics of Iris Melanocytic Tumors

PURPOSE To evaluate tumor vasculature with optical coherence tomography angiography (OCTA) in malignant iris melanomas and benign iris lesions. DESIGN Cross-sectional observational clinical study. PARTICIPANTS Patients with iris lesions and healthy volunteers. METHODS Eyes were imaged using OCTA systems operating at 1050- and 840-nm wavelengths. Three-dimensional OCTA scans were acquired. Iris melanoma patients treated with radiation therapy were imaged again after I-125 plaque brachytherapy at 6 and 18 months. MAIN OUTCOME MEASURES OCT and OCTA images, qualitative evaluation of iris and tumor vasculature, and quantitative vessel density. RESULTS One eye each of 8 normal volunteers and 9 patients with iris melanomas or benign iris lesions, including freckles, nevi, and an iris pigment epithelial (IPE) cyst, were imaged. The normal iris has radially oriented vessels within the stroma on OCTA. Penetration of flow signal in normal iris depended on iris color, with best penetration seen in light to moderately pigmented irides. Iris melanomas demonstrated tortuous and disorganized intratumoral vasculature. In 2 eyes with nevi there was no increased vascularity; in another, fine vascular loops were noted near an area of ectropion uveae. Iris freckles and the IPE cyst did not have intrinsic vascularity. The vessel density was significantly higher within iris melanomas (34.5%±9.8%, P < 0.05) than in benign iris nevi (8.0%±1.4%) or normal irides (8.0%±1.2%). Tumor regression after radiation therapy for melanomas was associated with decreased vessel density. OCTA at 1050 nm provided better visualization of tumor vasculature and penetration through thicker tumors than at 840 nm. But in very thick tumors and highly pigmented lesions even 1050-nm OCTA could not visualize their full thickness. Interpretable OCTA images were obtained in 82% of participants in whom imaging was attempted. CONCLUSIONS This is the first demonstration of OCTA in iris tumors. OCTA may provide a dye-free, no-injection, cost-effective method for monitoring a variety of tumors, including iris melanocytic lesions, for growth and vascularity. This could be helpful in evaluating tumors for malignant transformation and response to treatment. Penetration of the OCT beam remains a limitation for highly pigmented tumors, as does the inability to image the entire iris in a single field.

Uveal Melanoma with Histopathologic Intratumoral Heterogeneity Associated with Gene Expression Profile Discordance

Purpose: To report a case of intratumoral gene expression profile discordance in a malignant uveal melanoma, associated with intratumoral heterogeneity based upon histopathologic features. Methods: The clinical history, fundus findings, imaging and histopathologic features, and DecisionDx-UM gene expression profile results (Castle Biosciences, Inc., Phoenix, AZ, USA) of the tumor were reviewed. Results: A trans-retinal fine-needle aspiration biopsy was performed for a thin, pigmented choroidal tumor in a 33-year-old man. Cells obtained from this biopsy were tested using the DecisionDx-UM gene expression profile test and the tumor was classified as class 1A. Cytology confirmed melanoma. The patient subsequently elected to undergo enucleation. On microscopic examination of the globe, the tumor was composed primarily of spindle B cells, but had a focal area composed of epithelioid cells. This portion of the tumor was subsequently tested and demonstrated a class 1B gene expression profile. Conclusion: Intratumoral discordance in gene expression profile results has been described in uveal melanomas. Here we demonstrate that this discordance may be associated in some cases with intratumoral heterogeneity based upon histopathologic features.

Anterior uveitis associated with high-dose cytosine arabinoside.

Purpose: To report a case of anterior uveitis associated with high-dose cytosine arabinoside (Ara-C) treatment. Design: Observational case report. Methods: A 14-year-old girl was seen for pain, redness, and photophobia shortly after finishing a cycle of high-dose Ara-C. Results: Portable slit-lamp examination revealed bilateral diffuse fine keratic precipitates, indicative of a nongranulomatous anterior uveitis. This was treated successfully with a course of topical prednisolone acetate 1%. Conclusions: Ophthalmologists who are asked to evaluate patients on high-dose Ara-C for a “red eye” should consider anterior uveitis as a potential diagnosis.