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We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue. Microscopy is central to biological research and has enabled scientist to study the structure and dynamics of cells and their components within. Often, fluorescent dyes or trackers are used that can be detected under the microscope. However, this procedure can sometimes interfere with the biological processes being studied. Now, Guo, Yeh, Folkesson et al. have developed a new approach to examine structures within tissues and cells without the need for a fluorescent label. The technique, called QLIPP, uses the phase and polarization of the light passing through the sample to get information about its makeup. A computational model was used to decode the characteristics of the light and to provide information about the density and orientation of molecules in live cells and brain tissue samples of mice and human. This way, Guo et al. were able to reveal details that conventional microscopy would have missed. Then, a type of machine learning, known as ‘deep learning’, was used to translate the density and orientation images into fluorescence images, which enabled the researchers to predict specific structures in human brain tissue sections. QLIPP can be added as a module to a microscope and its software is available open source. Guo et al. hope that this approach can be used across many fields of biology, for example, to map the connectivity of nerve cells in the human brain or to identify how cells respond to infection. However, further work in automating other aspects, such as sample preparation and analysis, will be needed to realize the full benefits.

Background Glaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma. Methods Intracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Fas lpr ) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 μg/1 μl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR). Results Rebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Fas lpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1β, IL-6, IL-18, MIP-1α, MIP-1β, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3). Conclusions These results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity 1 – 3 . Changes to DNA methylation patterns over time form the basis of ageing clocks 4 , but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity 5 – 7 . Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1 ), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo. Expression of three Yamanaka transcription factors in mouse retinal ganglion cells restores youthful DNA methylation patterns, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice, suggesting that mammalian tissues retain a record of youthful epigenetic information that can be accessed to improve tissue function.

Fungal infections in humans occur as a result of defects in the immune system. An increasing emergence in oral Candidal and non-Candidal fungal infections is evident in the past decade owing to the rise in the immunodeficient and immunocompromised population globally. Oral Candidal infection usually involves a compromised host and the compromise may be local or systemic. Local compromising factors include decreased salivation, poor oral hygiene, wearing dentures among others while systemic factors include diabetes mellitus, nutritional deficiency, HIV infection/AIDS and others. Oral candidiasis is generally a localized infection and rarely appears as a systemic fungal disease whereas oral non-Candidal fungal infections are usually signs of disseminated disease. Some of the non-Candidal fungi that were once considered exotic and geographically restricted are now seen worldwide, beyond their natural habitat, probably attributed to globalization and travels. Currently infections from these fungi are more prevalent than before and they may present either as primary oral lesions or as oral manifestations of systemic mycoses. This review discusses the various predisposing factors, clinical presentations, clinical differential diagnosis, diagnosis and management of oral candidiasis, as well as briefly highlights upon a few of the more exotic non-Candidal fungi that infect the oral mucosa.

Ocular hypertension is a primary risk factor for glaucoma and results in retinal ganglion cell (RGC) degeneration. Current animal models of glaucoma lack severe RGC cell death as seen in glaucoma, making assessment of physiological mediators of cell death difficult. We developed a modified mouse model of ocular hypertension whereby long-lasting elevation of intraocular pressure (IOP) is achieved, resulting in significant reproducible damage to RGCs. In this model, microbeads are mixed with hyaluronic acid and injected into the anterior chamber of C57BL/6J mice. The hyaluronic acid allows for a gradual release of microbeads, resulting in sustained blockage of Schlemm's canal. IOP elevation was bimodal during the course of the model's progression. The first peak occurred 1 hours after beads injection, with an IOP value of 44.69 ± 6.00 mmHg, and the second peak occurred 6-12 days post-induction, with an IOP value of 34.91 ± 5.21 mmHg. RGC damage was most severe in the peripheral retina, with a loss of 64.1% compared to that of untreated eyes, while the midperiphery exhibited a 32.4% loss, 4 weeks following disease induction. These results suggest that sustained IOP elevation causes more RGC damage in the periphery than in the midperiphery of the retina. This model yields significant and reproducible RGC degeneration.

Age-related macular degeneration (AMD) is a chronic, progressive retinal disease characterized by an inflammatory response mediated by activated macrophages and microglia infiltrating the inner layer of the retina. In this study, we demonstrate that inhibition of macrophages through Siglec binding in the AMD eye can generate therapeutically useful effects. We show that Siglecs-7, -9 and -11 are upregulated in AMD associated M0 and M1 macrophages, and that these can be selectively targeted using polysialic acid (PolySia)-nanoparticles (NPs) to control dampen AMD-associated inflammation. In vitro studies showed that PolySia-NPs bind to macrophages through human Siglecs-7, -9, -11 as well as murine ortholog Siglec-E. Following treatment with PolySia-NPs, we observed that the PolySia-NPs bound and agonized the macrophage Siglecs resulting in a significant decrease in the secretion of IL-6, IL-1β, TNF-α and VEGF, and an increased secretion of IL-10. In vivo intravitreal (IVT) injection of PolySia-NPs was found to be well-tolerated and safe making it effective in preventing thinning of the retinal outer nuclear layer (ONL), inhibiting macrophage infiltration, and restoring electrophysiological retinal function in a model of bright light-induced retinal degeneration. In a clinically validated, laser-induced choroidal neovascularization (CNV) model of exudative AMD, PolySia-NPs reduced the size of neovascular lesions with associated reduction in macrophages. The PolySia-NPs described herein are therefore a promising therapeutic strategy for repolarizing pro-inflammatory macrophages to a more anti-inflammatory, non-angiogenic phenotype, which play a key role in the pathophysiology of non-exudative AMD.

Cells of the myeloid lineage are significant targets for human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in monkeys. Monocytes play critical roles in innate and adaptive immunity during inflammation. We hypothesize that specific subsets of monocytes expand with AIDS and drive central nervous system (CNS) disease. Additionally, there may be expansion of cells from the bone marrow through blood with subsequent macrophage accumulation in tissues driving pathogenesis. To identify monocytes that recently emigrated from bone marrow, we used 5-bromo-2'-deoxyuridine (BrdU) labeling in a longitudinal study of SIV-infected CD8+ T lymphocyte depleted macaques. Monocyte expansion and kinetics in blood was assessed and newly migrated monocyte/macrophages were identified within the CNS. Five animals developed rapid AIDS with differing severity of SIVE. The percentages of BrdU+ monocytes in these animals increased dramatically, early after infection, peaking at necropsy where the percentage of BrdU+ monocytes correlated with the severity of SIVE. Early analysis revealed changes in the percentages of BrdU+ monocytes between slow and rapid progressors as early as 8 days and consistently by 27 days post infection. Soluble CD163 (sCD163) in plasma correlated with the percentage of BrdU+ monocytes in blood, demonstrating a relationship between monocyte activation and expansion with disease. BrdU+ monocytes/macrophages were found within perivascular spaces and SIVE lesions. The majority (80-90%) of the BrdU+ cells were Mac387+ that were not productively infected. There was a minor population of CD68+BrdU+ cells (<10%), very few of which were infected (<1% of total BrdU+ cells). Our results suggest that an increased rate of monocyte recruitment from bone marrow into the blood correlates with rapid progression to AIDS, and the magnitude of BrdU+ monocytes correlates with the severity of SIVE.

A fourth generation poly-lysine dendritic nanocarrier (P4LDN)-based targeted chemotherapy for breast cancer is attempted by incorporating an epidermal growth factor receptor (EGFR)-specific short peptide E2 (ARSHVGYTGAR) and the drug methotrexate (MTX) into a nanocarrier system. The drug is incorporated into the nanocarrier using a cathepsin B cleavable spacer: glycine–phenylalanine–leucine–glycine (GFLG). The in vitro analysis of the time-dependent drug release, binding and internalization ability, and the cytotoxic nature showed that this drug delivery system (DDS) is highly effective. The efficacy analysis using non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice also showed that compared to the control group, the DDS can effectively reduce tumor volume. The mice that received the DDS appeared to gain weight more rapidly than the free drug, which suggests that the dendrimer is more easily tolerated by mice than the free drug.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

T2 lesion quantification plays a crucial role in monitoring disease progression and evaluating treatment response in multiple sclerosis (MS). We developed a 3D, multi-arm U-Net for T2 lesion segmentation, which was trained on a large, multicenter clinical trial dataset of relapsing MS. We investigated its generalization to other relapsing and primary progressive MS clinical trial datasets, and to an external dataset from the MICCAI 2016 MS lesion segmentation challenge. Additionally, we assessed the model’s ability to reproduce the separation of T2 lesion volumes between treatment and control arms; and the association of baseline T2 lesion volumes with clinical disability scores compared with manual lesion annotations. The trained model achieved a mean dice coefficient of ≥ 0.66 and a lesion detection sensitivity of ≥ 0.72 across the internal test datasets. On the external test dataset, the model achieved a mean dice coefficient of 0.62, which is comparable to 0.59 from the best model in the challenge, and a lesion detection sensitivity of 0.68. Lesion detection performance was reduced for smaller lesions (≤ 30 μL, 3–10 voxels). The model successfully maintained the separation of the longitudinal changes in T2 lesion volumes between the treatment and control arms. Such tools could facilitate semi-automated MS lesion quantification; and reduce rater burden in clinical trials.