The human splenic microcirculation is entirely open as shown by 3D models in virtual reality.

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.

Exploring human splenic red pulp vasculature in virtual reality: details of sheathed capillaries and the open capillary network.

We reconstructed serial sections of a representative adult human spleen to clarify the unknown arrangement of the splenic microvasculature, such as terminal arterioles, sheathed capillaries, the red pulp capillary network and venules. The resulting 3D model was evaluated in virtual reality (VR). Capillary sheaths often occurred after the second or third branching of a terminal arteriole and covered its capillary side or end branches. The sheaths started directly after the final smooth muscle cells of the arteriole and consisted of cuboidal CD271++ stromal sheath cells surrounded and infiltrated by B lymphocytes and macrophages. Some sheaths covered up to four sequential capillary bifurcations thus forming bizarre elongated structures. Each sheath had a unique form. Apart from symmetric dichotomous branchings inside the sheath, sheathed capillaries also gave off side branches, which crossed the sheath and freely ended at its surface. These side branches are likely to distribute materials from the incoming blood to sheath-associated B lymphocytes and macrophages and thus represent the first location for recognition of blood-borne antigens in the spleen. A few non-sheathed bypasses from terminal arterioles to the red pulp capillary network also exist. Red pulp venules are primarily supplied by sinuses, but they also exhibit a few connections to the capillary network. Thus, the human splenic red pulp harbors a primarily open microcirculation with a very minor closed part.

Plasma cells, plasmablasts, and AID+/CD30+ B lymphoblasts inside and outside germinal centres: details of the basal light zone and the outer zone in human palatine tonsils.

Plasma cells (PCs) in human palatine tonsils are predominantly located in the germinal centres (GCs), in the subepithelial space and near the deep connective tissue septa surrounding each crypt. We analysed the location, phenotype, and proliferation of GC PCs by immunohistology comparing them to PCs in the other two locations. Most PCs in GCs were strongly positive for CD38, CD138, CD27, IRF4, and intracellular (ic) IgG. They often accumulated in the basal light zone, but could also be found scattered in the entire light zone. In addition, rows of PCs occurred at the surface of the GC bordering the mantle zone, i.e., in the outer zone, and at the surface of the dark zone. The latter cells were often continuous with PCs in the extrafollicular area. The vast majority of GC PCs were negative for Ki-67. Only a few Ki-67+ plasmablasts, predominantly icIgG+ or icIgM+, were found inside GCs. In certain GCs PCs accumulated around capillaries and the adjacent perikarya of follicular dendritic cells (FDCs). Newly formed PCs might migrate from the basal to the superficial part of the light zone and then back to the dark zone surface to leave the GC. This guarantees an even distribution of secreted Ig for exchange with immune complexes on FDCs. The surface of the dark zone may also be an exit site for Ki-67+CD30+ B lymphoblasts, which seed perifollicular and extrafollicular sites. We speculate that these cells tend to downmodulate CD20 and activation-induced deaminase and further up-regulate CD30 when developing into pre-plasmablasts.

Recommendations of the working group of the Anatomische Gesellschaft on reduction of formaldehyde exposure in anatomical curricula and institutes.

The practice of human and veterinary medicine is based on the science of anatomy and dissection courses are still irreplaceable in the teaching of anatomy. Embalming is required to preserve body donors, for which process formaldehyde (FA) is the most frequently used and well characterized biocidal substance. Since January 2016, a new occupational exposure limit (OEL) for FA of 0.37mg/m3 issued by the European Committee on Hazardous Substances is obligatory since FA has been classified as a human 1B carcinogen. The anatomical institutes in the German-speaking region are called upon to consolidate efforts to reduce use of FA in anatomical curricula and body donations. As a result, the Anatomische Gesellschaft (AG) has formed a "Working Group for Reduction of Formaldehyde Exposure in Dissection Courses" tasked with discussion and recommendation of measures to reduce FA. Based on the assessment of the Working Group, the AG has issued an official opinion to the effect that, at this point in time, embalming of body donors without FA completely is not feasible. Therefore, a combination of approaches are to be used to reduce FA exposure, including technical and structural (architectural) adaptations, modification of protocols for fixation and preservation as well as organizational measures. One structural measure considered unavoidable is the integration of air supply and exhaust of individual dissecting tables into the ventilation system of the anatomy building. To embalm human body donors, intra-arterial perfusion fixation with up to 4% FA and a total fluid volume of 150mL/kg body weight will suffice. For animals where body weights and biology of bodies vary widely (i.e. special needs of fixation for ruminants, large animals as horses) perfusion fixation with up to 4% FA and a quantity of fixative solution of 10-15% of the body weight may be required. Preservation of body donors in storage (immersion) can be done with 40% ethanol or in a full bath preservation containing up to 2% FA. Corpse humidification in the dissecting room is possible with 2% phenoxyethanol, in each case without FA. In veterinary anatomy, microbiological burden is often higher and therefore might lead to a need of FA in long-time storage. Compliance with the current OEL in all institutes would appear to be feasible in combination with various organizational measures.

Capillary networks and follicular marginal zones in human spleens. Three-dimensional models based on immunostained serial sections.

We have reconstructed small parts of capillary networks in the human splenic white pulp using serial sections immunostained for CD34 alone or for CD34 and CD271. The three-dimensional (3D) models show three types of interconnected networks: a network with very few long capillaries inside the white pulp originating from central arteries, a denser network surrounding follicles plus periarterial T-cell regions and a network in the red pulp. Capillaries of the perifollicular network and the red pulp network have open ends. Perifollicular capillaries form an arrangement similar to a basketball net located in the outer marginal zone. The marginal zone is defined by MAdCAM-1+ marginal reticular stromal cells. Perifollicular capillaries are connected to red pulp capillaries surrounded by CD271+ stromal capillary sheath cells. The scarcity of capillaries inside the splenic white pulp is astonishing, as non-polarised germinal centres with proliferating B-cells occur in adult human spleens. We suggest that specialized stromal marginal reticular cells form a barrier inside the splenic marginal zone, which together with the scarcity of capillaries guarantees the maintenance of gradients necessary for positioning of migratory B- and T-lymphocytes in the human splenic white pulp.

Feature-based multi-resolution registration of immunostained serial sections.

The form and exact function of the blood vessel network in some human organs, like spleen and bone marrow, are still open research questions in medicine. In this paper, we propose a method to register the immunohistological stainings of serial sections of spleen and bone marrow specimens to enable the visualization and visual inspection of blood vessels. As these vary much in caliber, from mesoscopic (millimeter-range) to microscopic (few micrometers, comparable to a single erythrocyte), we need to utilize a multi-resolution approach. Our method is fully automatic; it is based on feature detection and sparse matching. We utilize a rigid alignment and then a non-rigid deformation, iteratively dealing with increasingly smaller features. Our tool pipeline can already deal with series of complete scans at extremely high resolution, up to 620 megapixels. The improvement presented increases the range of represented details up to smallest capillaries. This paper provides details on the multi-resolution non-rigid registration approach we use. Our application is novel in the way the alignment and subsequent deformations are computed (using features, i.e. "sparse"). The deformations are based on all images in the stack ("global"). We also present volume renderings and a 3D reconstruction of the vascular network in human spleen and bone marrow on a level not possible before. Our registration makes easy tracking of even smallest blood vessels possible, thus granting experts a better comprehension. A quantitative evaluation of our method and related state of the art approaches with seven different quality measures shows the efficiency of our method. We also provide z-profiles and enlarged volume renderings from three different registrations for visual inspection.

Three-Dimensional Arrangement of Human Bone Marrow Microvessels Revealed by Immunohistology in Undecalcified Sections.

The arrangement of microvessels in human bone marrow is so far unknown. We combined monoclonal antibodies against CD34 and against CD141 to visualise all microvessel endothelia in 21 serial sections of about 1 cm2 size derived from a human iliac crest. The specimen was not decalcified and embedded in Technovit® 9100. In different regions of interest, the microvasculature was reconstructed in three dimensions using automatic methods. The three-dimensional models were subject to a rigid semiautomatic and manual quality control. In iliac crest bone marrow, the adipose tissue harbours irregularly distributed haematopoietic areas. These are fed by networks of large sinuses, which are loosely connected to networks of small capillaries prevailing in areas of pure adipose tissue. Our findings are compatible with the hypothesis that capillaries and sinuses in human iliac crest bone marrow are partially arranged in parallel.

Human spleen microanatomy: why mice do not suffice.

The microanatomical structure of the spleen has been primarily described in mice and rats. This leads to terminological problems with respect to humans and their species-specific splenic microstructure. In mice, rats and humans the spleen consists of the white pulp embedded in the red pulp. In the white pulp, T and B lymphocytes form accumulations, the periarteriolar lymphatic sheaths and the follicles, located around intermediate-sized arterial vessels, the central arteries. The red pulp is a reticular connective tissue containing all types of blood cells. The spleen of mice and rats exhibits an additional well-delineated B-cell compartment, the marginal zone, between white and red pulp. This area is, however, absent in human spleen. Human splenic secondary follicles comprise three zones: a germinal centre, a mantle zone and a superficial zone. In humans, arterioles and sheathed capillaries in the red pulp are surrounded by lymphocytes, especially by B cells. Human sheathed capillaries are related to the splenic ellipsoids of most other vertebrates. Such vessels are lacking in rats or mice, which form an evolutionary exception. Capillary sheaths are composed of endothelial cells, pericytes, special stromal sheath cells, macrophages and B lymphocytes. Human spleens most probably host a totally open circulation system, as connections from capillaries to sinuses were not found in the red pulp. Three stromal cell types of different phenotype and location occur in the human white pulp. Splenic white and red pulp structure is reviewed in rats, mice and humans to encourage further investigations on lymphocyte recirculation through the spleen.

Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells, follicular dendritic cells and a third superficial stromal cell type.

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD2711- and CD271+/) stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27+ and switched B lymphocytes, but also scattered IgD++ B lymphocytes and variable numbers of CD4+ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far.

B lymphocyte compartments in the human splenic red pulp: capillary sheaths and periarteriolar regions.

The microvasculature of human spleens is still incompletely understood. Two enigmatic types of red pulp microvessels, penicillar arterioles and sheathed capillaries, have already been described in the nineteenth century without gaining much attention afterwards. We performed a detailed analysis of sheathed capillaries to clarify the cellular composition of their sheaths by immunohistological double-staining experiments. Capillary sheaths comprise three different cell types, namely specialized cuboidal CD271(++) inner sheath cells surrounded by CD271(-) macrophages and accumulations of B lymphocytes. The CD271(++) inner sheath cells express the chemokine CXCL13 in a unique single dot pattern. Sheath-associated B lymphocytes consist of IgM(+), IgD(++), and of "switched" cells. T lymphocytes do not accumulate in pericapillary sheaths. The predominant sheath-associated macrophage population is CD163(-)CD68(+) and thus differs from the majority of red pulp macrophages. The sheath-associated macrophages strongly express CD169 only in perifollicular sheaths, but not in sheaths located deeper in the red pulp. IgM(+), IgD(++), and "switched" B cells are also closely associated with red pulp arterioles characterized by the expression of smooth muscle actin in muscle cells and in branched periarteriolar stromal cells. Capillary sheaths are observed in a post-arteriolar position and appear to be of limited length. We suggest to change the term "Vagina periarteriolaris makrophagocytica" of the international histological and embryological terminologies to "Vagina pericapillaris."

Immunostaining of pulpal nerve fibre bundle/arteriole associations in ground serial sections of whole human teeth embedded in technovit® 9100.

A technique for embedding human undecalcified tooth specimens in Technovit® 9100 was developed, which permits immunohistological evaluation of pulp tissue in serial ground sections. Human molars were divided into 14-18 sections of about 23 µm thickness. Immunohistological double staining for S-100 and CD34 revealed unique associations of myelinated nerve fibre bundles with arterioles, which continued through the entire tooth pulp. These arterioles were not only accompanied by, but partially or totally enveloped in longitudinally orientated myelinated nerve fibre bundles. We speculate that this unique arrangement may mechanically support the arterioles and alleviate detection or regulation of their contraction state by sensory nerve cells.

The open microcirculation in human spleens: a three-dimensional approach.

It has long been debated whether the red pulp of human spleens harbors an open or a closed microcirculation or both. To solve this issue, the authors differentially stained the endothelium in red pulp arterial microvessels and in venous sinuses using brightfield and fluorescence immunohistology with reagents against CD34 and CD141. Three-dimensional models of red pulp arterial microvessels and sinuses were derived from serial double-stained paraffin sections with the help of license-free open-access software. In each model, arterial microvascular ends were traced and verified by reference to the original serial sections. In total, 142 ends were analyzed in the specimens of three individuals. None of these ends was connected to a sinus, suggesting that the human splenic red pulp harbors an entirely open circulatory system. Thus, the spleen is the only human organ where blood passes through spaces not lined by endothelia or other barrier-forming cells.

The follicular dendritic cell network in secondary follicles of human palatine tonsils and spleens.

The structure of germinal centres (GCs) in human secondary lymphatic organs has not been thoroughly investigated until now. We stained follicular dendritic cells (FDCs) in serial sections of human hyperplastic tonsils and spleens to compare the morphology of GCs in fulminant immune reactions and quiescence. Detection of CD35, CD21, CD23 and the target of mAb CNA.42 confirmed that full-blown human tonsil GCs may consist of four regions, the dark zone, the basal and apical light zone and the outer zone. The outer zone was, however, not a constant feature of tonsillar GCs and existed only in a minority of follicles in most specimens. Thus, between 3 and 60% of tonsil GCs with a CD23(+) apical light zone exhibited an outer zone in individual specimens. FDCs in tonsil GCs appeared to be extremely sensitive to mechanical stress during surgery. In contrast to tonsils, seven of the eight adult spleens did not exhibit asymmetric polarized GCs, but only symmetric GCs without dark and light zones or follicles with few GC B cells. Some specimens apparently only contained primary follicles after conventional staining, but on closer inspection a homogenous hyaline extracellular material deposited among the FDCs indicated that a GC had been present. Our study demonstrates that the structure of GCs varies in different human secondary lymphatic organs most likely depending on the local antigenic challenge.

CD1d expression in paneth cells and rat exocrine pancreas revealed by novel monoclonal antibodies which differentially affect NKT cell activation.

BACKGROUND: CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated. METHODOLOGY/PRINCIPAL FINDINGS: Two mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb. CONCLUSIONS/SIGNIFICANCE: The two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d(-/-) mice, which resulted in an altered composition of the gut flora.

Donor B cells in splenic follicles of experimental pulmonary allograft recipients.

BACKGROUND: After transplantation, passenger leukocytes move to lymphoid organs of the recipient. These cells appear to initiate allograft rejection, but they also might be involved in tolerance induction. MATERIALS AND METHODS: Orthotopic left lung transplantation was performed in the Dark Agouti to Lewis rat strain combination with no immunosuppression. Recipient spleens were removed at intervals of 24 h until day 6 after transplantation. For comparison, spleens from renal allograft recipients were analysed. Donor-derived major histocompatibility complex (MHC) class II antigens were detected by monoclonal antibody OX76. In double-staining experiments with antibodies specific for leukocyte subpopulations, their localisation and identity was analysed. RESULTS: OX76-positive leukocytes were already detected in recipient spleens on day 1 post-transplantation. They increased in number until day 3 and decreased in number thereafter. Most of them were localised in splenic follicles and expressed the B cell variant of CD45R and IgG. Cell surface antigens typical for other leukocyte subpopulations were not detected. In the spleens of renal allograft recipients, only few donor-derived cells were seen. CONCLUSION: After lung transplantation, numerous MHC class II-positive B cells migrate to the splenic follicles of the recipient. These cells might, in part, be responsible for immunologic differences observed between renal and pulmonary allografts.

Phenotypic differences between red pulp capillary and sinusoidal endothelia help localizing the open splenic circulation in humans.

The distribution of capillaries, sinuses and larger vessels was investigated by immunohistology in paraffin sections of 12 adult human spleens using a panel of antibodies. Double staining for CD34 and CD141 (thrombomodulin) revealed that capillary endothelia in the cords of the splenic red pulp and at the surface of follicles were CD34(+)CD141(-), while red pulp sinus endothelia had the phenotype CD34(-)CD141(+). Only in the direct vicinity of splenic follicles did sinus endothelial cells exhibit both antigens. Thus, splenic sinuses do not replace conventional capillaries, but exist in addition to such vessels. The endothelium in arterioles, venules and larger arteries and veins was uniformly CD34(+)CD141(+). Anti-CD34 and anti-CD141 both additionally reacted with different types of splenic stromal cells. Differential staining of capillaries and sinuses may permit a three-dimensional reconstruction of serial sections to unequivocally delineate the "open" and "closed" splenic circulation in humans.

Fetal and early post-natal development of the human spleen: from primordial arterial B cell lobules to a non-segmented organ.

Immunohistological analysis of 31 human spleens from the 11th week of gestation to the early postnatal period suggested that fetal organ development may be preliminarily divided into four stages. At stage 0 the organ anlage contained erythrocyte precursors, few macrophages and almost no lymphocytes. Fetal spleens of stage I exhibited arterial vascular lobules and lymphocytes just began colonizing the organ. At stage II, B and T lymphocytes formed periarteriolar clusters. B cell clusters predominated, because B cells aggregated around the more peripheral branches of splenic arterioles, while T cells occupied the more centrally located parts of the vessels. The vascular lobules of stage I and II consisted of central arterioles surrounded by B cells, capillaries and peripheral venules. The lobular architecture slowly dissolved at late stage II when sinuses grew out from the peripheral venules into the centre of the lobule. Interestingly, the B cell accumulations around peripheral arterioles did not represent the precursors of follicles, but apparently persisted as periarteriolar B cell clusters in the adult splenic red pulp, while follicles containing FDCs developed at late stage II from B cells in direct contact to T cell clusters around larger arterial vessels. At stage III before birth the lobular architecture was no longer recognized. The chemokine CXCL13 was already present in vascular smooth muscle and adjacent stromal cells at stage I before B cells immigrated. CCL21, on the contrary, was only demonstrated in fibroblast-like cells supporting T cell clusters from stage II onwards.

The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants.

The role of the spleen in B memory cell development and maintenance is attracting increased attention. Studies in mice and rats have indicated that memory functions are associated with large B cells residing in the marginal zone (MZ) of the spleen. Although the cellular composition of the MZ is relatively well known in these species, controversies exist about the function of MZ B cells, their dependence on the presence of the spleen and the stage at which their development branches from that of recirculating follicular B cells. Additional confusion has arisen with respect to MZ B cells in humans, because the microscopic anatomy of the human splenic MZ differs decisively from that of rodents. Several recent publications indicate that the functional and migratory properties of human MZ B cells may be species-specific. The hypothesis derived from these publications and from our immunohistological observations implies that at least a major number of human splenic CD27(+) MZ B cells are migratory. Phenotypic data suggest a recirculation pathway between the spleen and mucosal tissues in humans.

CD27+ B cells in human lymphatic organs: re-evaluating the splenic marginal zone.

The marginal zone of human spleens is regarded as an organ-specific region harbouring sessile memory B cells. This opinion has arisen by extrapolating from results obtained in mice and rats. Detection of CD27(+) B cells in situ now revealed similarities among the most superficial region of B-cell follicles in human spleens, reactive lymph nodes, inflamed appendices, tonsils and terminal ilea. The follicular surface in these organs consists of small naïve immunoglobulin D (IgD)(+) CD27(-) B cells predominating in an inner area and larger IgD(+/-) CD27(+) B cells prevailing in a more superficial position. CD27(+) B cells may, however, also occupy the entire follicular periphery around the germinal centre. Together with additional peculiarities this distribution indicates a fundamental microanatomical difference among the human and rodent splenic white pulp. We hypothesize that the follicular periphery represents a recirculation compartment both for naïve and memory/natural reactive B cells in all human secondary lymphatic organs. This assumption implies a difference in recirculation behaviour among human and rodent B memory cells.

Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire.

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive, immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) "memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis, complementarity-determining region 3 (CDR3) spectratyping during a T-independent response, and gene-expression profiling of the different blood and splenic B-cell subsets, we show here that blood IgM(+)IgD(+)CD27(+) cells correspond to circulating splenic marginal zone B cells. Furthermore, analysis of this peripheral subset in healthy children younger than 2 years shows that these B cells develop and mutate their immunoglobulin receptor during ontogeny, prior to their differentiation into T-independent antigen-responsive cells. It is therefore proposed that these IgM(+)IgD(+)CD27(+) B cells provide the splenic marginal zone with a diversified and protective preimmune repertoire in charge of the responses against encapsulated bacteria.