A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations

Design and Analysis of Rhesus Cytomegalovirus IL-10 Mutants as a Model for Novel Vaccines against Human Cytomegalovirus

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection

Comparison of Antimetabolite Drugs as Corticosteroid-Sparing Therapy for Noninfectious Ocular Inflammation

To compare the relative effectiveness and side effect profiles of antimetabolite drugs in the treatment of noninfectious ocular inflammation. Retrospective cohort study. A total of 257 patients with inflammatory eye disease seen in a single-center, academic practice and treated with an antimetabolite as a first-line immunosuppressive agent from 1984 to 2006. Data recorded included demographics, antimetabolite and prednisone doses, use of other immunosuppressive drugs, response to therapy, and side effects associated with drug use. Ability to control ocular inflammation and to taper prednisone to ≤10 mg daily (“treatment success”); incidence of treatment-related side effects. Ninety patients with inflammatory eye disease were treated with methotrexate, 38 patients were treated with azathioprine, and 129 patients were treated with mycophenolate. Uveitis accounted for the majority of the diagnoses (67%, 66%, and 68% for methotrexate, azathioprine, and mycophenolate, respectively), followed by scleritis (23%, 18%, 17% for methotrexate, azathioprine, and mycophenolate, respectively). The median time to treatment success was 4.0, 4.8, and 6.5 months for the mycophenolate, azathioprine, and methotrexate treatment groups, respectively ( P = 0.02, log-rank test). The incidence of side effects was higher in the azathioprine group (0.29/person-year [PY]) compared with patients treated with methotrexate (0.14/PY) and mycophenolate (0.18/PY). More patients discontinued the drug because of side effects in the azathioprine group (0.24/PY vs 0.09/PY for the methotrexate group and 0.09/PY for the mycophenolate mofetil group). These data suggest that the time to control of ocular inflammation is faster with mycophenolate than with methotrexate. Azathioprine therapy has a higher rate of treatment-related side effects compared with the other 2 agents. The authors have no proprietary or commercial interest in any materials discussed in this article

Quantifying Color Vision Changes Associated With Cataracts Using Cone Contrast Thresholds.

PurposeTo evaluate effects of age and simulated and real cataractous changes on color vision as measured by the high-definition cone contrast test (CCT).MethodsTwenty-four healthy volunteers from two cohort studies performed CCT using best-corrected visual acuity, filters, mydriasis, and pinhole correction. Retrospective cross-sectional study of patients seen in eye clinics evaluated the relationship between age and color vision, and age and lens status in 355 eyes. Last, 25 subjects underwent CCT before and after cataract surgery.ResultsCCT scores were most reliable in the nonmydriatic condition without pinhole correction. Progressively dense brown filters produced small decreases in S-cone sensitivity. Linear regression analysis of phakic subjects showed a decline for all cone classes with age. Rate of decline was greater for S-cones (slope = -1.09; 95% confidence interval [CI], -1.30 to 0.86) than M-cones (slope = -0.80; 95% CI, -1.03 to -0.58) and L-cones (slope = -0.66; 95% CI, -0.88 to -0.44). CCT scores increased for S-cones but reduced for L- and M-cones in pseudophakic subjects compared with phakic patients. CCT scores after cataract surgery increased for S-cones, M-cones, and L-cones by 33.0 (95% CI, 8.6 to 57.4), 24.9 (95% CI, 3.8 to 46.0), and 22.0 (95% CI, -3.2 to 47.3), respectively.ConclusionsCCT assessment allows for clinically practical quantitation of color and contrast vision improvement after cataract surgery and aging patients who note poor vision despite good visual acuity.Translational relevanceCCT testing, which quantifies hereditary and acquired color deficiency, can also quantify the degree of cataract severity and, combined with other parameters, can provide more precise guidance for cataract extraction to optimize patient care