PURPOSE . To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence. METHODS . Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation. RESULTS . Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double -membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years. CONCLUSIONS . We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

Hypoxic ischemic encephalopathy (HIE) occurs in 2-5/1000 births, with acute kidney injury (AKI) occurring in 40%. AKI increases morbidity and mortality. Caffeine, an adenosine receptor antagonist, and photobiomodulation (PBM), working on cytochrome c oxidase, are potential treatments for AKI. To examine effects of caffeine and PBM on AKI in rats, Day 7 pups underwent a HIE intervention (Modified Rice-Vannucci model) replicating pathology observed in humans. Caffeine was administered for 3 days and/or PBM for 5 days following HIE. Weights and urine for biomarkers (NGAL, albumin, KIM-1, osteopontin) were collected prior to HIE, daily post intervention and at sacrifice. Both treatments reduced kidney injury seen on electron microscopy, but not when combined. HIE elevated urinary NGAL and albumin on Days 1-3 post-HIE, before returning to control levels. This elevation was significantly reduced by PBM or caffeine. KIM-1 was significantly elevated for 7 days post-HIE and was reduced by both treatments. Osteopontin was not altered by HIE or the treatments. Treatments, individually but not in combination, improved HIE-induced reductions in the enzymatic activity of mitochondrial complexes II-III. PBM and caffeine also improved weight gain. PBM and caffeine reduces AKI diagnosed by urinary biomarkers and confirmed by EM findings.

Hypoxic ischemic encephalopathy (HIE) is associated with acute kidney injury (AKI) in neonates with birth asphyxia. This study aimed to utilize urinary biomarkers to characterize AKI in an established neonatal rat model of HIE. Day 7 Sprague-Dawley rat pups underwent HIE using the Rice-Vannucci model (unilateral carotid ligation followed by 120 mins of 8% oxygen). Controls included no surgery and sham surgery. Weights and urine for biomarkers (NGAL, osteopontin, KIM-1, albumin) were collected the day prior, daily for 3days post-intervention, and at sacrifice day 14. Kidneys and brains were processed for histology. HIE pups displayed histological evidence of kidney injury including damage to the proximal tubules, consistent with resolving acute tubular necrosis, and had significantly elevated urinary levels of NGAL and albumin compared to sham or controls 1-day post-insult that elevated for 3days. KIM-1 significantly increased for 2days post-HIE. HIE did not significantly alter osteopon tin levels. Seven days post-start of experiment, controls were 81.2% above starting weight compared to 52.1% in HIE pups. NGAL and albumin levels inversely correlated with body weight following HIE injury. The AKI produced by the Rice-Vannucci HIE model is detectable by urinary biomarkers, which can be used for future studies of treatments to reduce kidney injury.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease 1 – 7 , evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA 8 , 9 and binds to guanosine 10 – 12 . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP 10 – 12 , and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c + age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7 Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Pancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states. Mice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate. Deletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response. These findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.

We identify an intronic deletion in VANGL1 that predisposes to renal injury in high risk populations through a kidney-intrinsic process. Half of all SLE patients develop nephritis, yet the predisposing mechanisms to kidney damage remain poorly understood. There is limited evidence of genetic contribution to specific organ involvement in SLE.(1,2) We identify a large deletion in intron 7 of Van Gogh Like 1 (VANGL1), which associates with nephritis in SLE patients. The same deletion occurs at increased frequency in an indigenous population (Tiwi Islanders) with 10-fold higher rates of kidney disease compared with non-indigenous populations. Vangl1 hemizygosity in mice results in spontaneous IgA and IgG deposition within the glomerular mesangium in the absence of autoimmune nephritis. Serum transfer into B cell-deficient Vangl1(+/-) mice results in mesangial IgG deposition indicating that Ig deposits occur in a kidney-intrinsic fashion in the absence of Vangl1. These results suggest that Vangl1 acts in the kidney to prevent Ig deposits and its deficiency may trigger nephritis in individuals with SLE.

Background: Glioblastoma Multiforme (GBM) is an aggressive brain cancer associated with high mortality. Current treatments including resection of 98% or more of the tumour result in median survival of less than 15 months. With such poor outcomes, innovative therapies are required. Ultrasmall Superparamagnetic Iron Oxide Nanoparticles (USPIONs) are emerging as potential treatments, however little is known on their effects on cells. Understanding interactions between USPIONs and GBM is critical in developing these therapies. The aim of this study is to investigate biological effects of USPION uptake on GBM cells in vitro. Methods: CNS-1 cell cultures were exposed to 20µg/mL of USPIONs with a maghemite iron oxide cores, which mean core diameters are 10–15 nm. In order to measure the intake and the interaction of USPIONs on CNS-1, we have utilized different techniques including the transmission electron microscopy (TEM), iron quantification, mitochondrial membrane potential assay, Oxidative Stress Test, Mitochondrial Transition Pore Assay, flow cytometry, immunohistochemistry and western blotting. Results: Our results showed that USPIONs entered CNS-1 cells via clatherin coated pits which then became internalized in vacuoles. USPIONs induced Fenton Reaction, which potentially leads to the oxidative stress activating the Heat Shock Proteins (HSPs) protective mechanism. When this mechanism was overwhelmed, it led to a decrease in cell viability, however in due course, cells upregulated HSPs, re-activating these protective mechanisms which included the closure of mitochondrial permeability transition pore, limiting the release of pro-apoptotic cytochrome c, reducing oxidative stress and eventually recovering cell viability. Conclusions: Ultrasmall superparamagnetic iron oxide nanoparticles interact with CNS-1, initiating the mitochondrial death pathway, however heat shock proteins are recruited, mitigating further apoptosis. Targeting CNS-1 protective mechanisms in conjunction with USPIONs exposure could induce a cytotoxic effect on CNS-1, providing insights for a novel therapy for this devastating disease. Legal entity responsible for the study: Tailoi Chan-Ling Funding: None Disclosure: All authors have declared no conflicts of interest.

Introduction: Fibrillary glomerulonephritis is characterised by the glomerular deposition of randomly arranged non branching fibrils which differ from amyloid fibrils in size (typically 16-24nm in diameter as compared with 7–10 nm for amyloid) and lack of reactivity with congo red histochemistry. Immunotactoid glomerulopathy, widely but not universally considered distinct from fibrillary glomerulonephritis, is distinguished by deposition of immunoglobulin organised as microtubules, which typically measure >30 nm diameter. The diagnosis of each relies on electron microscopic identification of the glomerular deposits. Case report: A 77 year old woman was admitted for investigation of nephrotic syndrome (proteinuria 3.1 g/24 hours). A renal biopsy showed a mesangiocapillary glomerulonephritis, IgM positivity on direct immunofluorescence and lambda light chains, and trace positivity with C1q. There was no positivity with IgG, IgA, C3 and kappa light chains. Electron microscopy showed glomerular deposition of microtubules with a diameter of 34 nm. She was found to have an IgM lambda paraprotein and diagnosed with IgM MGUS. Discussion: Immunotactoid glomerulopathy is a rare entity occurring one tenth as frequently as fibrillary glomerulonephritis,1 tends to occur in older patients, who typically present with nephrotic syndrome, and is often secondary to an underlying cause such as a lymphoplasmacytic disorder. The most common light microscopic pattern is mesangiocapillary, and direct immunofluorescence is typically positive for IgG, with variable positivity of the other immunoreactants. As seen in our case, IgM positivity is unusual, and is not reported without concurrent IgG positivity. Reference1.Rosenstock JL1, Markowitz GS, Valeri AM. Fibrillary and immunotactoid glomerulonephritis: Distinct entities with different clinical and pathologic features. Kidney Int 2003; 63: 1450–61.

PURPOSE Lymphatics subserve many important functions in the human body including maintenance of fluid homeostasis, immune surveillance, and tumor metastasis. Our aim was to provide structural and phenotypic evidence of lymphatic-like structures in the human choroid, including details of its development. METHODS Using multiple-marker immunohistochemistry (IHC), choroids from human fetal eyes (8-26 weeks gestation) and adults (17-74 years) were examined with lymphatic- and vascular-specific markers: prospero homeobox-1 (PROX-1), lymphatic vascular endothelium receptor-1 (LYVE-1), podoplanin, D2-40, endomucin, VEGF-C, vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4), UEA lectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), CD34, and CD39. Transmission electron microscopy (TEM) was used to establish evidence for choroidal lymphatics, and to provide details of stratification and relative frequency of lymphatics compared to choroidal blood vessels. RESULTS Immunohistochemistry and TEM indicated a central-to-peripheral topography of lymphatic formation, with numerous blind-ended lymph sacs just external to the choriocapillaris, as well as the presence of infrequent precollector and collector lymphatic channels. Characteristic ultrastructural features of lymphatics in adult human choroid included anchoring filaments, luminal flocculent protein but absence of erythrocytes, fragmented and/or absent basal lamina, absence of intracellular Weibel-Palade bodies, infrequent pericyte ensheathment, and lack of fenestrae. CONCLUSIONS The system of blind-ended initial lymphatic segments seen just external to the fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid and strategically placed for immune surveillance. The presence of a system of lymphatic-like channels in the human choroid provides an anatomical basis for antigen presentation in the posterior eye, with a possible route from the eye to the sentinel lymph nodes, similar to that already described for anterior eye lymphatics.

We read with interest the recent article by Koina et al.1 suggesting evidence for the presence of lymphatic vessels in the developing and adult human choroid. However, this study does not meet the recently published consensus criteria on the immunohistochemical detection of ocular lymphatic vessels,2 and therefore, in our opinion, requires critical revision. First, appropriate positive and unequivocal negative controls are not presented in the study of Koina et al. In particular, when describing novel anatomical structures for the first time, and in order to change an existing dogma, a detailed documentation of blood and lymphatic vessel detection in the control tissue is mandatory. The provided supplementary data do not fulfill these criteria. Second, the immunohistochemical marker panel used is critical. Endomucin does not represent an established lymphatic marker,3,4 but is rather expressed by “endothelial cells along the whole vascular tree including lymphatic vessels.”5 Thus, an unequivocal discrimination between blood and lymphatic vessels is impossible with this marker. A further discrepancy is the use of the transcription factor prospero-related homebox gene-1 (Prox-1) as an extranuclear lymphatic endothelial precursor marker. Although reports of the extranuclear presence of PROX-1 in cell types other than lymphatic endothelium exist,6–8 PROX-1 clearly shows a nuclear expression in lymphatic endothelia in human,9 as well as mouse10 and avian,11 embryos, retaining its nuclear localization into adulthood.12–14 On the other hand, it is not clear why lymphatic endothelial surface markers, such as podoplanin, lymphatic vascular endothelial-specific hyaluronic acid receptor-1 (LYVE-1), and the vascular endothelial marker CD34 display nuclear expression in this study. Additionally, the only lymphatic endothelial cell marker used in whole mounts is VEGFR-3, which is also expressed in fenestrated blood vessels, and, as such, also in the choriocapillaris.15,16 Morphologically, the supposed lymphatic VEGFR-3–positive vessels are indistinguishable from the honeycomb-like lobular pattern of the choriocapillaris.17 Furthermore, the study of Koina et al. includes a blatant inconsistency in the use and documentation of immunohistochemical markers between fetal and adult eyes. Although one has to acknowledge that certain lymphatic markers might be expressed during embryogenesis, this pattern easily changes during maturation.18 Therefore, such an approach would require extensive comparison of the same markers in different ages, thus representing an extensive survey in its own right. However, this is not the case in the study of Koina et al. Third, the ultrastructural study would be greatly strengthened by immunoelectron microscopy. Indeed, anchoring filaments with a diameter of 40 to 100 A—becoming readily identifiable only at magnifications of 40,000× to 50,000×—are present in lymphatics,19 but could be easily present in the choroid as well without any association to lymphatic vessels,20–22 particularly in aged eyes with typical alterations of the extracellular matrix. For this purpose, as well as for ruling out Weibel-Palade bodies, serial ultrathin sectioning with appropriate labeling would be necessary. Despite possible postmortem tissue alterations, numerous previous studies successfully applied different detection systems for ultrastructural investigations using ocular human donor tissue.23–29 A limited use of immunomarkers for these investigations, as claimed, seems therefore not justified. In regard to the above-mentioned criticisms, the evidence presented in the study of Koina et al. does not justify the hypothesized paradigm shift that functional lymphatic vessels are present in the human choroid. Rather, the findings of Koina et al. confirm previous reports of net-like structures with a “pseudo-vessel” appearance in the human choroid endowed with lymphatic vascular precursor cells (represented as LYVE-1+ macrophages).25 Those “atypical” lymphatic-like cells (i.e., endothelial cells with divergent or uncommon immunohistochemical phenotypes) may also exist in other parts of the eye. For example, the endothelial cells of Schlemm's canal display many, but not all, features of terminally differentiated lymphatic endothelial cells, including responsiveness to VEGF-C–induced lymphangiogenesis.30 In closing, we acknowledge that the work of Koina et al. is a further contribution to our understanding of the choroid, but although the existence of lymphatics in the human choroid cannot be ruled out per se, because of the aforementioned points and the sheer volume of evidence to date, we maintain that the inner human eye and in particular the choroid should still be considered an immune-privileged site devoid of lymphatic vessels. Further unequivocal evidence of “typical lymphatic vessels” in the human choroid is still missing.

BACKGROUND & AIMS:Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury. METHODS:In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4). RESULTS:In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 μM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 μM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes. CONCLUSIONS:These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.

Indomethacin and ibuprofen are administered to close a patent ductus arteriosus (PDA) during active glomerulogenesis. Light and electron microscopic glomerular changes with no change in glomerular number were seen following indomethacin and ibuprofen treatment during glomerulogenesis at 14 days after birth in a neonatal rat model. This present study aimed to determine whether longstanding renal structural changes are present at 30 days and 6 mo (equivalent to human adulthood). Rat pups were administered indomethacin or ibuprofen antenatally on days 18–20 (0.5 mg·kg −1 ·dose −1 indomethacin; 10 mg·kg −1 ·dose −1 ibuprofen) or postnatally intraperitoneally from day 1 to 3 or day 1 to 5 (0.2 mg·kg −1 ·dose −1 indomethacin; 10 mg·kg −1 ·dose −1 ibuprofen). Control groups received no treatment or normal saline intraperitoneally. Pups were killed at 30 days of age and 6 mo of age. Tissue blocks from right kidneys were prepared for light and electron microscopic examination, while total glomerular number was determined in left kidneys using unbiased stereology. Eight pups were included in each group from 14 maternal rats. At 30 days and 6 mo, there were persistent electron microscopy abnormalities of the glomerular basement membrane in those receiving postnatal indomethacin and ibuprofen. There were no significant light microscopy findings at 30 days or 6 mo. At 6 mo, there were significantly fewer glomeruli in those receiving postnatal indomethacin but not ibuprofen ( P = 0.003). In conclusion, indomethacin administered during glomerulogenesis appears to reduce the number of glomeruli in adulthood. Alternative options for closing a PDA should be considered including ibuprofen as well as emerging therapies such as paracetamol.