Chronic kidney disease affects 1 in 10 people worldwide, with damage to specialized blood filter cells of the kidney, called podocytes, playing a critical role. In membranous nephropathy (MN), a major cause of nephrotic syndrome, circulating autoantibodies attack proteins on podocyte foot processes (FPs), damaging the kidney's filtration barrier. Our study shows that these autoantibodies trigger the formation of antigen-autoantibody aggregates on the podocyte FP plasma membrane. These aggregates bud off as stalked vesicles, termed autoimmunoglobulin-triggered extracellular vesicles (AIT-EVs), which are released into the urine. AIT-EVs carry disease-causing autoantibodies, their target antigens, essential FP proteins, and disease-associated stressors representing a mechanism for removing immune complexes (ICs) and waste. However, their excessive release leads to FP effacement and podocyte dysfunction. In MN patients, urinary AIT-EVs correspond to glomerular urinary-space aggregates. Enriching AIT-EVs enables detection and monitoring of pathogenic autoantibodies, suggesting a non-invasive approach for autoimmune kidney disease diagnosis and therapy.

ABSTRACTBackgroundMembranous nephropathy (MN) is caused by autoantibody binding to podocyte foot process antigens such as THSD7A and PLA2R1. The mechanisms of the glomerular antigen/autoantibody deposition and clearance are unknown.MethodsWe explore the origin and significance of glomerular accumulations in (1) diagnostic and follow-up biospecimens from THSD7A+and PLA2R1+-MN patients compared to nephrotic non-MN patients, and (2) in experimental models of THSD7A+-MN.ResultsWe discovered podocyte exophers as correlates of histological antigen/autoantibody aggregates found in the glomerular urinary space of MN patients. Exopher vesicle formation represents a novel form of toxic protein aggregate removal inCaenorhabditis elegansneurons. In MN patients, podocytes released exophers to the urine. Enrichment of exophers from MN patient urines established them as a glomerular exit route for antigens and bound autoantibody. Exophers also carried disease-associated proteins such as complement and provided a molecular imprint of podocyte injury pathways. In experimental THSD7A+-MN, exophers were formed from podocyte processes and cell body. Their formation involved the translocation of antigen/autoantibody from the subepithelial to the urinary side of podocyte plasma membranes. Urinary exopher-release correlated with lower albuminuria and lower glomerular antigen/autoantibody burden. In MN patients the prospective monitoring of urinary exopher abundance and of exopher-bound autoantibodies was additive in the assessment of immunologic MN activity.ConclusionsExopher-formation and release is a novel pathomechanism in MN to remove antigen/autoantibody aggregates from the podocyte. Tracking exopher-release will add a non-invasive diagnostic tool with prognostic potential to clinical diagnostics and follow-up of MN patients.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography—mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell–cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell–cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Purpose: To date, there are many theories about tear transport through the canaliculi of the draining lacrimal system into the lacrimal sac but only few with supportive data. It is certain that the function of the lacrimal part of orbicularis oculi muscle (Horner-Duverney's muscle) is indispensable for the transport of "used" tears. However, the muscle's exact structure and the mechanisms of its functions are as yet unclear. To obtain deeper insights we undertook the present study. Methods: Upper and lower canaliculi (including the entrance into the lacrimal sac) from donor cadavers were dissected. Some of the specimens were prepared for scanning electron microscopy (SEM) to analyze the course of muscle fibers surrounding the canaliculi. Others were sectioned for enzyme(EHC) and immunohistochemistry (IHC) to learn about the distribution of slow and fast reacting muscle fibers in Horner-Duverney's muscle as well as to analyze the distribution of different neurotransmitters to learn more about the innervation of Horner-Duverney's muscle. Four tear duct systems taken from body donors were cut out en bloc after formalin fixation, serially sectioned and reconstructed using a newly developed technology for 3D reconstruction of histological serial sections named HiD (R) (Chimaera GmbH, Germany). Patients that had undergone dacryocystorhinostomy (DCR) were video-analyzed endonasally during active blinking, focusing on viewing the temporal wall of the lacrimal sac movement where the canaliculi penetrated the lacrimal sac. Results: SEM revealed that muscle fibers of Horner-Duverney's muscle surround the vertical parts of the upper and lower canaliculus in a scissor like pattern whereas they ran in parallel to the first two thirds of the horizontal parts surrounding the respective canaliculus. Here, the muscle fibers were embedded in dense connective tissue forming a unique network. At the nasal third, muscle fibers left the canaliculi and ran to the posterior part of the fascia of the lacrimal sac and the lacrimal bone. EHC revealed that Horner-Duverney's muscle contained nearly an equal distribution of type I and type IIb muscle fibers compared to the superior rectus muscle which contains more type I and the masseter and iliopsoas muscles with more type IIb muscle fibers. IHC indicated presence of trigeminal, catecholaminergic and cholinergic nerve endings. 3D reconstructions supported the SEM data. Endonasal video analysis of patients after DCR with a nasally open lacrimal sac revealed bulging of the temporal wall of the lacrimal sac during blinking. On the basis of these findings, a modified lacrimal pump theory is proposed. Conclusion: The results support the hypothesis that contraction of Horner-Duverney's muscle leads to closure of the canaliculi in their first two thirds based on the special arrangement of muscle fibers and connective tissue fibers. This causes the tear fluid in the canaliculi to be pressed/transported towards the lacrimal sac. The medial third of the vertical portions of the canaliculi, the canaliculus communis and the intrasaccal portion of the canaliculus are compressed by the shortening and thickening of the Horner-Duverney muscle from dorsal, which leads to a compression of the canaliculi lumens in this part of the system, thereby pushing the lacrimal fluid further towards the lacrimal sac. The mix of fast contracting and fatigue resistant muscle fibers is ideally suited for the blink mechanism that is complexly regulated by the nervous system.

The human spleen is equipped with an organ-specific microcirculation. The initial part of the venous circulation is formed by spleen-specific large microvessels, the sinuses. Sinuses eventually fuse to form venules and veins. For more than 170 years there have been debates, whether splenic red pulp capillaries join sinuses, i.e., whether the microcirculation is closed or open—or even simultaneously closed and open. We have now solved this question by three-dimensional reconstruction of a limited number of immunostained serial sections of red and white pulp areas, which were visualized in virtual reality. Splenic capillaries have special end structures exhibiting multiple small diverging endothelial cell processes, which always keep a certain distance to the walls of sinuses. Only very few capillary ends were difficult to diagnose. Positive identification of these end structures permits to conclude that the human splenic microcirculation is entirely open. This is also true for the perifollicular capillary network and for capillaries close to red pulp venules. Follicles are supplied by a relatively dense open perifollicular capillary net, which is primarily, but not exclusively, fed by sheathed and few non-sheathed capillaries from the surrounding red pulp network.

Registration of histological serial sections is a challenging task. Serial sections exhibit distortions and damage from sectioning. Missing information on how the tissue looked before cutting makes a realistic validation of 2D registrations extremely difficult. This work proposes methods for ground-truth-based evaluation of registrations. Firstly, we present a methodology to generate test data for registrations. We distort an innately registered image stack in the manner similar to the cutting distortion of serial sections. Test cases are generated from existing 3D data sets, thus the ground truth is known. Secondly, our test case generation premises evaluation of the registrations with known ground truths. Our methodology for such an evaluation technique distinguishes this work from other approaches. Both under- and over-registration become evident in our evaluations. We also survey existing validation efforts. We present a full-series evaluation across six different registration methods applied to our distorted 3D data sets of animal lungs. Our distorted and ground truth data sets are made publicly available.

The snack food potato chips induces food intake in ad libitum fed rats, which is associated with modulation of the brain reward system and other circuits. Here, we show that food intake in satiated rats is triggered by an optimal fat/carbohydrate ratio. Like potato chips, an isocaloric fat/carbohydrate mixture influenced whole brain activity pattern of rats, affecting circuits related e.g. to reward/addiction, but the number of modulated areas and the extent of modulation was lower compared to the snack food itself.

Three-dimensional (3-D) reconstruction of histological slice sequences offers great benefits in the investigation of different morphologies. It features very high-resolution which is still unmatched by in vivo 3-D imaging modalities, and tissue staining further enhances visibility and contrast. One important step during reconstruction is the reversal of slice deformations introduced during histological slice preparation, a process also called image unwarping. Most methods use an external reference, or rely on conservative stopping criteria during the unwarping optimization to prevent straightening of naturally curved morphology. Our approach shows that the problem of unwarping is based on the superposition of low-frequency anatomy and high-frequency errors. We present an iterative scheme that transfers the ideas of the Gauss-Seidel method to image stacks to separate the anatomy from the deformation. In particular, the scheme is universally applicable without restriction to a specific unwarping method, and uses no external reference. The deformation artifacts are effectively reduced in the resulting histology volumes, while the natural curvature of the anatomy is preserved. The validity of our method is shown on synthetic data, simulated histology data using a CT data set and real histology data. In the case of the simulated histology where the ground truth was known, the mean Target Registration Error (TRE) between the unwarped and original volume could be reduced to less than 1 pixel on average after six iterations of our proposed method.

In this paper we present a super-resolving approach for detecting an axially moving target that is based upon a time-multiplexing concept and that overcomes the diffraction limit set by the optics of an imaging camera by a priori knowledge of the high-resolution background in front of which the target is moving. As the movement trajectory is axial, the approach can be applied to targets that are approaching or moving away from the camera. By recording a set of low-resolution images at different target axial positions, the super-resolving algorithm weights each image by demultiplexing them using the high-resolution background image and provides a super-resolved image of the target. Theoretical analyses as well as simulations and preliminary experimental validation are presented to validate the proposed approach.

Non-homeostatic hyperphagia, which is a major contributor to obesity-related hyperalimentation, is associated with the diet's molecular composition influencing, for example, the energy content. Thus, specific food items such as snack food may induce food intake independent from the state of satiety. To elucidate mechanisms how snack food may induce non-homeostatic food intake, it was tested if manganese-enhanced magnetic resonance imaging (MEMRI) was suitable for mapping the whole brain activity related to standard and snack food intake under normal behavioral situation. Application of the MnCl2 solution by osmotic pumps ensured that food intake was not significantly affected by the treatment. After z-score normalization and a non-affine three-dimensional registration to a rat brain atlas, significantly different grey values of 80 predefined brain structures were recorded in ad libitum fed rats after the intake of potato chips compared to standard chow at the group level. Ten of these areas had previously been connected to food intake, in particular to hyperphagia (e.g. dorsomedial hypothalamus or the anterior paraventricular thalamic nucleus) or to the satiety system (e.g. arcuate hypothalamic nucleus or solitary tract); 27 areas were related to reward/addiction including the core and shell of the nucleus accumbens, the ventral pallidum and the ventral striatum (caudate and putamen). Eleven areas associated to sleep displayed significantly reduced Mn2+-accumulation and six areas related to locomotor activity showed significantly increased Mn2+-accumulation after the intake of potato chips. The latter changes were associated with an observed significantly higher locomotor activity. Osmotic pump-assisted MEMRI proved to be a promising technique for functional mapping of whole brain activity patterns associated to nutritional intake under normal behavior.

In this study, we present first data concerning the anatomical structure, blood supply and location of the lacrimal gland of the pig. Our data indicate that the porcine lacrimal gland may serve as a potential xenograft candidate in humans or as an animal model for engineering of a bioartificial lacrimal gland tissue construct for clinical application. For this purpose, we used different macroscopic preparation techniques and digital reconstruction of the histological gland morphology to gain new insights and important information concerning the feasibility of a lacrimal gland transplantation from pig to humans in general. Our results show that the lacrimal gland of the pig reveals a lot of morphological similarities to the analogous human lacrimal gland and thus might be regarded as a xenograft in the future. This is true for a similar anatomical location within the orbit as well as for the feeding artery supply to the organ. Functional differences concerning the composition of the tear fluid, due to a different secretory unit distribution within the gland tissue will, however, be a challenge in future investigations.

The inspection of histological image sequences to gain knowledge about the original three-dimensional (3-D) morphological structure is a standard method in medical research. Its main advantage is that light microscopes feature high resolution enhanced visibility due to staining. In many cases this imaging technology could immensely profit from 3-D reconstructions of the slice images. For volumetric stacking, however, the tissue deformations due to slice preparation require an unwarping strategy to restore the original morphology. The challenge is to reverse the artificial deformations while preserving the natural morphological changes. In particular, unintentional straightening of curved structures across multiple slices has to be avoided. In this article, we propose a novel way to incorporate landmarks representing the morphological progression. They are used as additional regularization for intensity based non-rigid registration which is capable to exactly match the landmarks. Our approach is tested on synthetical and histological data sets. We show that it delivers smooth contours while preserving the morphological structure, and is a promising addition to existing methods.

It is a common approach to create and inspect histological slices to investigate functional and morphological structures on a cellular level. For the easier analysis of the resulting data sets, the underlying 3-D structure has to be reconstructed and visualized. Due to mechanical stress imposed on the tissue during the slicing, some slices are damaged, leading to an unsatisfying reconstruction result. In this article we present a means to interpolate missing images. For this, a deformation field, calculated by a variational non-rigid registration method using adjacent slices as reference and template images, is partially applied to the template image. The approach is tested on a histological data set, and evaluated using visual inspection by experts. The resulting reconstruction is shown to be a significant improvement.