Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation.

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

Synergistic Chromatin-Modifying Treatments Reactivate Latent HIV and Decrease Migration of Multiple Host-Cell Types.

Upon infection of its host cell, human immunodeficiency virus (HIV) establishes a quiescent and non-productive state capable of spontaneous reactivation. Diverse cell types harboring the provirus form a latent reservoir, constituting a major obstacle to curing HIV. Here, we investigate the effects of latency reversal agents (LRAs) in an HIV-infected THP-1 monocyte cell line in vitro. We demonstrate that leading drug treatments synergize activation of the HIV long terminal repeat (LTR) promoter. We establish a latency model in THP-1 monocytes using a replication incompetent HIV reporter vector with functional Tat, and show that chromatin modifiers synergize with a potent transcriptional activator to enhance HIV reactivation, similar to T-cells. Furthermore, leading reactivation cocktails are shown to differentially affect latency reactivation and surface expression of chemokine receptor type 4 (CXCR4), leading to altered host cell migration. This study investigates the effect of chromatin-modifying LRA treatments on HIV latent reactivation and cell migration in monocytes. As previously reported in T-cells, epigenetic mechanisms in monocytes contribute to controlling the relationship between latent reactivation and cell migration. Ultimately, advanced "Shock and Kill" therapy needs to successfully target and account for all host cell types represented in a complex and composite latency milieu.

A photoswitchable GPCR-based opsin for presynaptic inhibition.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Peripapillary Retinoschisis in Glaucoma: Association With Progression and OCT Signs of Müller Cell Involvement.

Purpose: To examine demographic and clinical factors associated with glaucomatous peripapillary retinoschisis (PPRS) and assess its association with glaucoma progression. Methods: Using a case control study design and longitudinal data from a cohort of 166 subjects with a diagnosis of glaucoma or glaucoma suspect, we compared functional, structural, clinical, and demographic characteristics between PPRS cases and controls. Results: The frequency of PPRS was 6.0% (12 eyes from 10/166 subjects) with two eyes having PPRS in different sectors for a total of 15 retinoschisis events. There were no significant differences (P > 0.05) in age, sex, visual acuity, central corneal thickness, intraocular pressure, or presence of vitreous adhesion between PPRS and controls. However, eyes with PPRS tended to have a higher cup-to-disc ratio (P = 0.06), thinner RNFL (P = 0.02), and worse visual field mean deviation (MD, P = 0.06) than controls. The rate of RNFL thinning was faster in PPRS (average: -2.8%/year; range: -7.4% to 0.0%/year) than controls (-1.3%/year; range: -4.4% to 0.6%/year; P = 0.021). The rate of visual field MD change was faster in PPRS (-0.49 dB/year; range: -2.0 to 0.9 dB/year) than controls (-0.06 dB/year; range: -0.8 to 0.3 dB/year; P = 0.030). OCT scans showed hyperreflective structures spanning the PPRS whose morphology and spacing (50 ± 7 µm) are consistent with Müller glia, causing signal attenuation casting "shadows" onto distal retina. Conclusions: This is the first report showing that glaucomatous PPRS is associated with a faster overall rate of RNFL thinning and visual field deterioration and to specifically identify OCT signs of Müller cell involvement.

Projection-Resolved Optical Coherence Tomography Angiography of Macular Retinal Circulation in Glaucoma.

PURPOSE: To detect macular perfusion defects in glaucoma using projection-resolved optical coherence tomography (OCT) angiography. DESIGN: Prospective observation study. PARTICIPANTS: A total of 30 perimetric glaucoma and 30 age-matched normal participants were included. METHODS: One eye of each participant was imaged using 6×6-mm macular OCT angiography (OCTA) scan pattern by 70-kHz 840-nm spectral-domain OCT. Flow signal was calculated by the split-spectrum amplitude-decorrelation angiography algorithm. A projection-resolved OCTA (PR-OCTA) algorithm was used to remove flow projection artifacts. Four en face OCTA slabs were analyzed: the superficial vascular complex (SVC), intermediate capillary plexus (ICP), deep capillary plexus (DCP), and all-plexus retina (SVC + ICP + DCP). The vessel density (VD), defined as the percentage area occupied by flow pixels, was calculated from en face OCTA. A novel algorithm was used to adjust the vessel density to compensate for local variations in OCT signal strength. MAIN OUTCOME MEASURES: Macular retinal VD, ganglion cell complex (GCC) thickness, and visual field (VF) sensitivity. RESULTS: Focal capillary dropout could be visualized in the SVC, but not the ICP and DVP, in glaucomatous eyes. In the glaucoma group, the SVC and all-plexus retinal VD (mean ± standard deviation: 47.2%±7.1% and 73.5%±6.6%) were lower than in the normal group (60.5%±4.0% and 83.2%±4.2%, both P < 0.001, t test). The ICP and DCP VD were not significantly lower in the glaucoma group. Among the overall macular VD parameters, the SVC VD had the best diagnostic accuracy as measured by the area under the receiver operating characteristic curve (AROC). The accuracy was even better when the worse hemisphere (inferior or superior) was used, achieving an AROC of 0.983 and a sensitivity of 96.7% at a specificity of 95%. Among the glaucoma participants, the hemispheric SVC VD values were highly correlated with the corresponding GCC thickness and VF sensitivity (P < 0.003). The reflectance compensation step in VD calculation significantly improved repeatability, normal population variation, and correlation with VF and GCC thickness. CONCLUSIONS: On the basis of PR-OCTA, glaucoma preferentially affects perfusion in the SVC in the macula more than the deeper plexuses. Reflectance-compensated SVC VD measurement by PR-OCTA detected glaucoma with high accuracy and could be useful in the clinical evaluation of glaucoma.

A New Variant of Polypoidal Choroidal Vasculopathy With Annular Pigmentary Changes in Haitian Males.

The authors report a new variant of idiopathic polypoidal choroidal vasculopathy (IPCV) in middle-aged Haitian men characterized by extramacular polypoidal lesions and bilateral extensive pigmentary alterations in the posterior pole in an annular wreath-like pattern surrounding the optic nerve and macular area. Two patients were seen at Massachusetts Eye and Ear Infirmary and one at Boston University Medical Center between 2010 and 2015. All three patients were middle-aged Haitian men who exhibited bilateral features of IPCV, including subretinal hemorrhages and serosanguinous pigment epithelial detachments. Indocyanine green angiography revealed extramacular polypoidal lesions located mostly along the major vascular arcades. Extensive pigmentary alterations were evident in the posterior pole surrounding the macula and optic nerve in an annular wreath-like pattern. These cases further expand the clinical spectrum of IPCV.

A Novel Technique for Amniotic Membrane Transplantation in Patients with Acute Stevens-Johnson Syndrome.

Cryopreserved amniotic membrane (AM) transplantation is an emerging technique that is becoming the gold standard for the management of acute Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN). We describe a novel surgical technique utilizing a single, large sheet of AM (5 x 10 cm) and a custom-made forniceal ring, which facilitates AM placement. Our technique is easy to use and minimizes suturing and manipulation of ocular tissues, resulting in decreased operative time. This technique may be applied in the management of multiple ocular surface disease processes, including chemical or thermal burns, severe ocular graft versus host disease (GVHD), and other autoimmune diseases.

Vomiting of oral medications by pediatric patients: survey of medication redosing practices.

BACKGROUND: At the time of this study, the authors' pediatric tertiary care hospital had no policy to guide actions when a child vomited after ingesting oral medication, and limited information was available in the literature. OBJECTIVES: To characterize this clinical problem at the study hospital, to identify current practices related to redosing of medications at the study hospital, and to collect guidelines and opinions of health care professionals at other pediatric hospitals on this topic. METHODS: Two online surveys were conducted, each over a 1-month period in late 2010, to identify current practices and opinions of pediatric health care professionals about redosing of medications after vomiting. E-mail distribution lists and health care forums were used to recruit participants. RESULTS: Of the 76 responses from the study hospital, 65 were suitable for analysis. Many respondents reported encountering vomiting after administration of oral medications on a weekly (25 [38%]) or monthly (24 [37%]) basis. Most of the respondents reported that they would follow a general rule to redose if vomiting occurred within 30 min (39 [60%]) or 15 min (21 [32%]) after initial ingestion. When respondents were asked to rate the importance of 8 factors potentially affecting the decision to redose, more than half indicated that time after dose ingestion (59 [91%]), medication type (45 [69%]), patient status (39 [60%]), and visibility of medication in the vomitus (36 [55%]) were very important. Of the 53 respondents to the survey of health care professionals at other institutions, 16 (30%) indicated that their pediatric hospital or ward had a guideline on redosing in cases of vomiting after administration of oral medications. Most respondents (12/13 [92%]) stated that the guideline took into account the interval between initial ingestion and vomiting. CONCLUSIONS: The problem of vomiting after administration of an oral medication was prevalent at the study hospital, and guidelines were scarce at other pediatric institutions. Health care professionals at the study hospital and other institutions listed the time between ingestion and vomiting as the most important factor in the decision to redose the medication.

Decay accelerating factor (CD55)-mediated attenuation of complement: therapeutic implications for age-related macular degeneration.

PURPOSE: Sequence variations in complement proteins are associated with age-related macular degeneration (AMD). The terminal pathway of complement results in the formation of the membrane attack complex (MAC) on the cell surface, resulting in their lysis. MAC has been documented on the retinal pigment epithelium (RPE), choroidal blood vessels, and drusen of AMD eyes. Here the investigators test the hypothesis that increasing the expression of decay accelerating factor (CD55) on RPE cells may result in reduced MAC-mediated damage. METHODS: The investigators constructed a recombinant adenovirus expressing human CD55 (AdCAGCD55). Mouse hepatocytes were infected with AdCAGCD55 or negative controls and subsequently incubated with normal human serum (NHS). Cell lysis and MAC formation were measured by FACS and immunocytochemistry, respectively. Adult mice were injected in the subretinal space with either AdCAGCD55 or controls; after 1 week of CD55 transgene expression, the eyecups were excised, challenged with NHS, and quantified for human MAC formation. RESULTS: Control-infected or uninfected mouse hepatocytes lyse at a rate of 93% and 94%, respectively. AdCAGCD55- infected mouse hepatocytes lyse at a rate of 29%. Lysis was confirmed to occur in the presence of MAC, which was reduced by 67% when cells were infected by AdCAGCD55. Mice injected in the subretinal space with AdCAGCD55 exhibited a 55.7% reduction in MAC formation on the RPE relative to controls. CONCLUSIONS: Adenovirus-mediated delivery of hCD55 to murine RPE confers protection against human complement. The investigators propose that the expression of hCD55 on RPE cells warrants investigation as a potential therapy for AMD.