The role of desmoglein-2 in kidney disease.

Desmosomes are multi-protein cell-cell adhesion structures supporting cell stability and mechanical stress resilience of tissues, best described in skin and heart. The kidney is exposed to various mechanical stimuli and stress, yet little is known about kidney desmosomes. In healthy kidneys, we found desmosomal proteins located at the apical-junctional complex in tubular epithelial cells. In four different animal models and patient biopsies with various kidney diseases, desmosomal components were significantly upregulated and partly miss-localized outside of the apical-junctional complexes along the whole lateral tubular epithelial cell membrane. The most upregulated component was desmoglein-2 (Dsg2). Mice with constitutive tubular epithelial cell-specific deletion of Dsg2 developed normally, and other desmosomal components were not altered in these mice. When challenged with different types of tubular epithelial cell injury (unilateral ureteral obstruction, ischemia-reperfusion, and 2,8-dihydroxyadenine crystal nephropathy), we found increased tubular epithelial cell apoptosis, proliferation, tubular atrophy, and inflammation compared to wild-type mice in all models and time points. In vitro, silencing DSG2 via siRNA weakened cell-cell adhesion in HK-2 cells and increased cell death. Thus, our data show a prominent upregulation of desmosomal components in tubular cells across species and diseases and suggest a protective role of Dsg2 against various injurious stimuli.

Reduction of Endothelial Glycocalyx on Peritubular Capillaries in Chronic Kidney Disease.

In chronic kidney disease (CKD), peritubular capillaries undergo anatomic and functional alterations, such as rarefaction and increased permeability. The endothelial glycocalyx (EG) is a carbohydrate-rich gel-like mesh, which covers the luminal surface of endothelial cells. It is involved in many regulatory functions of the endothelium, including vascular permeability. Herein, we investigated ultrastructural alterations of the EG in different murine CKD models. Fluorescence staining using different lectins with high affinity to components of the renal glycocalyx revealed a reduced binding to the endothelium in CKD in the animal models, and there were similar finding in human kidney specimens. Lanthanum Dysprosium Glycosamino Glycan adhesion staining technique was used to visualize the ultrastructure of the glycocalyx in transmission electron microscopy. This also enabled quantitative analyses, showing a significant reduction of the EG thickness and density. In addition, mRNA expression of proteins involved in glycocalyx biology, synthesis, and turnover (ie, syndecan 1 and glypican 1), which are main components of the glycocalyx, and exostosin 2, involved in the synthesis of the glycocalyx, were significantly up-regulated in endothelial cells isolated from murine CKD models. Visualization of glycocalyx using specific transmission electron microscopy analyses allows qualitative and quantitative analyses and revealed significant pathologic alterations in peritubular capillaries in CKD.

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

Anaerobic single-cell dispensing facilitates the cultivation of human gut bacteria.

Cultivation via classical agar plate (CAP) approaches is widely used to study microbial communities, but they are time-consuming. An alternative approach is the application of single-cell dispensing (SCD), which allows high-throughput, label-free sorting of microscopic particles. We aimed to develop a new anaerobic SCD workflow to cultivate human gut bacteria and compared it with CAP using faecal communities on three rich culture media. We found that the SCD approach significantly decreased the experimental time to obtain pure cultures from 17 ± 4 to 5 ± 0 days, while the isolate diversity and relative abundance coverage were comparable for both approaches. We further tested the total captured fraction by sequencing the sorted bacteria directly after growth as bulk biomass from 2400 dispensed single cells without downstream identification of individual strains. In this approach, the cultured fraction increased from 35.2% to 52.2% for SCD, highlighting the potential for deeper cultivation projects from single samples. SCD-based cultivation also captured species not detected by sequencing (16 ± 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes and Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera and 10 novel species that were fully characterized taxonomically.

Platelet count reduction during in vitro membrane oxygenation affects platelet activation, neutrophil extracellular trap formation and clot stability, but does not prevent clotting.

INTRODUCTION: Due to improved technology and increased application the mortality during extracorporeal membrane oxygenation (ECMO) is constantly declining. Nevertheless, complications including haemorrhage or thrombus formation remain frequent. Local mitigation of coagulation within an ECMO system to prevent thrombus formation on ECMO components and optimizing systemic anticoagulation is an approach to reduce clotting and bleeding complications at once. Foreign surfaces of ECMO systems, activate platelets (PLTs), which besides their major role in coagulation, can trigger the formation of neutrophil extracellular traps (NETs) contributing to robust thrombus formation. The impact of a reduced PLT count on PLT activation and NET formation is of paramount importance and worth investigating. METHODS: In this study platelet poor (PLT-) and native (PLT+) heparinized human blood was circulated in two identical in vitro test circuits for ECMO devices for 6 hours. PLT reduction was achieved by a centrifugation protocol prior to the experiments. To achieve native coagulation characteristics within the test circuits, the initial heparin dose was antagonized by continuous protamine administration. RESULTS: The PLT- group showed significantly lower platelet activation, basal NET formation and limited clot stability measured via thromboelastometry. Fluorescent and scanning electron microscope imaging showed differences in clot composition. Both groups showed equal clot formation within the circuit. CONCLUSIONS: This study demonstrated that the reduction of PLTs within an ECMO system is associated with limited PLT activation and NET formation, which reduces clot stability but is not sufficient to inhibit clot formation per se.

Effects of Perfusion Pressures on Podocyte Loss in the Isolated Perfused Mouse Kidney.

BACKGROUND/AIMS: Podocytes are lost in most glomerular diseases, leading to glomerulosclerosis and progressive kidney disease. It is generally assumed, that podocytes are exposed to the filtration flow and thus to significant shear forces driving their detachment from the glomerular basement membrane (GBM). In this context, foot process effacement has been proposed as potential adaptive response to increase adhesion of podocytes to the GBM. METHODS: We have tested these hypotheses using optical clearing and high-resolution 3-dimensional morphometric analysis in the isolated perfused murine kidney. We investigated the dynamics of podocyte detachment at different perfusion pressures (50, 300 and more than 450 mmHg) in healthy young or old mice (20 vs. 71 weeks of age), or mice injected with anti-GBM serum to induce global foot process effacement. RESULTS: Results show that healthy podocytes in young mice are tightly attached onto the GBM and even supramaximal pressures did not cause significant detachment. Compared to young mice, in aged mice and mice with anti-GBM nephritis and foot process effacement, gradual progressive loss of podocytes had occurred already before perfusion. High perfusion pressures resulted in a relatively minor additional loss of podocytes in aged mice. In mice with anti-GBM nephritis significant additional podocyte loss occurred at this early time point when increasing perfusion pressures to 300 mmHg or higher. CONCLUSION: This work provides the first experimental evidence that podocytes are extraordinarily resistant to acutely increased perfusion pressures in an ex vivo isolated kidney perfusion model. Only in glomerular disease, significant numbers of injured podocytes detached following acute increases in perfusion pressure.

Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension.

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

Modeling of magnetoliposome uptake in human pancreatic tumor cells in vitro.

The internalization kinetics resulting from magnetic nanoparticle interactions with tumor cells play an important role in nanoparticle-based cancer treatment efficiency. Here, the uptake kinetics of magnetoliposomes (ML) into human pancreatic tumor cells (MiaPaCa-2 and BxPC-3) are quantified using magnetic particle spectrometry. A comparison to the uptake kinetics for healthy L929 cells is given. The experimental results are used for the development of an uptake kinetics model describing the three relevant internalization processes: ML adsorption to the cell membrane, endo- and exocytosis. By fitting of experimental data, the rate constant of each internalization process is determined enabling the prediction of internalized ML at any incubation time. After seven hours incubation time, MiaPaCa-2 internalized three times more ML than BxPC-3 and L929 cells even though their ML adsorption rate constants were nearly the same. As the interaction of the ML with the cell membrane is non-specific, the uptake kinetics mirror the individual cell response to ML internalization. With a new mathematical term to cover the exocytosis contribution to the overall internalization process, the extended uptake kinetics model offers new possibilities to analyze the specific internalization mechanism for other nanoparticle and cell types.

Macrophage Migration Inhibitory Factor Limits Renal Inflammation and Fibrosis by Counteracting Tubular Cell Cycle Arrest.

Renal fibrosis is a common underlying process of progressive kidney diseases. We investigated the role of macrophage migration inhibitory factor (MIF), a pleiotropic proinflammatory cytokine, in this process. In mice subjected to unilateral ureteral obstruction, genetic deletion or pharmacologic inhibition of MIF aggravated fibrosis and inflammation, whereas treatment with recombinant MIF was beneficial, even in established fibrosis. In two other models of progressive kidney disease, global Mif deletion or MIF inhibition also worsened fibrosis and inflammation and associated with worse kidney function. Renal MIF expression was reduced in tubular cells in fibrotic compared with healthy murine and human kidneys. Bone marrow chimeras showed that Mif expression in bone marrow-derived cells did not affect fibrosis and inflammation after UUO. However, Mif gene deletion restricted to renal tubular epithelial cells aggravated these effects. In LPS-stimulated tubular cell cultures, Mif deletion led to enhanced G2/M cell-cycle arrest and increased expression of the CDK inhibitor 1B (p27Kip1) and of proinflammatory and profibrotic mediators. Furthermore, MIF inhibition reduced tubular cell proliferation in vitro In all three in vivo models, global Mif deletion or MIF inhibition caused similar effects and attenuated the expression of cyclin B1 in tubular cells. Mif deletion also resulted in reduced tubular cell apoptosis after UUO. Recombinant MIF exerted opposing effects on tubular cells in vitro and in vivo Our data identify renal tubular MIF as an endogenous renoprotective factor in progressive kidney diseases, raising the possibility of pharmacologic intervention with MIF pathway agonists, which are in advanced preclinical development.

Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries.

Progressive renal diseases are associated with rarefaction of peritubular capillaries, but the ultrastructural and functional alterations of the microvasculature are not well described. To study this, we analyzed different time points during progressive kidney damage and fibrosis in 3 murine models of different disease etiologies. These models were unilateral ureteral obstruction, unilateral ischemia-reperfusion injury, and Col4a3-deficient mice, we analyzed ultrastructural alterations in patient biopsy specimens. Compared with kidneys of healthy mice, we found a significant and progressive reduction of peritubular capillaries in all models analyzed. Ultrastructurally, compared with the kidneys of control mice, focal widening of the subendothelial space and higher numbers of endothelial vacuoles and caveolae were found in fibrotic kidneys. Quantitative analysis showed that peritubular capillary endothelial cells in fibrotic kidneys had significantly and progressively reduced numbers of fenestrations and increased thickness of the cell soma and lamina densa of the capillary basement membrane. Similar ultrastructural changes were also observed in patient's kidney biopsy specimens. Compared with healthy murine kidneys, fibrotic kidneys had significantly increased extravasation of Evans blue dye in all 3 models. The extravasation could be visualized using 2-photon microscopy in real time in living animals and was mainly localized to capillary branching points. Finally, fibrotic kidneys in all models exhibited a significantly greater degree of interstitial deposition of fibrinogen. Thus, peritubular capillaries undergo significant ultrastructural and functional alterations during experimental progressive renal diseases, independent of the underlying injury. Analyses of these alterations could provide read-outs for the evaluation of therapeutic approaches targeting the renal microvasculature.

The role of PDGF-D in healthy and fibrotic kidneys.

Platelet-derived growth factor (PDGF)-D, a specific PDGF receptor ß (PDGFR-ß) ligand, mediates mesangial proliferation in vitro and in vivo. However, its role in renal development, physiology, and fibrosis is relatively unknown. In healthy murine kidneys, PDGF-D was found to be expressed on renal mesenchymal cells (mesangial cells, fibroblasts, and vascular smooth muscle cells). During renal fibrosis, PDGF-D and its receptor PDGFR-ß were markedly and similarly upregulated in both human and murine kidneys on activated mesenchymal cells, but PDGF-D was also expressed de novo in injured renal tubular cells. The functional role of PDGF-D was studied in Pdgfd-/- mice, which showed no obvious spontaneous renal phenotype at a young age or during aging. Compared with wild-type littermates, Pdgfd-/- mice had significantly reduced renal interstitial fibrosis in two models of renal scarring: unilateral ureteral obstruction and unilateral ischemia/reperfusion injury. This was associated with reduced phosphorylation of PDGFR-ß and its downstream mediator p38. Systemic adenoviral overexpression of PDGF-D in healthy mice resulted in increased collagen deposition in the kidney interstitium. Thus, PDGF-D is upregulated in murine and human kidney fibrosis, may mediate renal scarring, and is dispensable for normal kidney development and physiological functions. PDGF-D may be a suitable therapeutic target to combat kidney fibrosis.

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities.

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria, and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Early-Life Immune System Maturation in Chickens Using a Synthetic Community of Cultured Gut Bacteria.

The gut microbiome is crucial for both maturation of the immune system and colonization resistance against enteric pathogens. Although chicken are important domesticated animals, the impact of their gut microbiome on the immune system is understudied. Therefore, we investigated the effect of microbiome-based interventions on host mucosal immune responses. Increased levels of IgA and IgY were observed in chickens exposed to maternal feces after hatching compared with strict hygienic conditions. This was accompanied by increased gut bacterial diversity as assessed by 16S rRNA gene amplicon sequencing. Cultivation work allowed the establishment of a collection of 43 bacterial species spanning 4 phyla and 19 families, including the first cultured members of 3 novel genera and 4 novel species that were taxonomically described. This resource is available at www.dsmz.de/chibac A synthetic community consisting of nine phylogenetically diverse and dominant species from this collection was designed and found to be moderately efficient in boosting immunoglobulin levels when provided to chickens early in life.IMPORTANCE The immune system plays a crucial role in sustaining animal health. Its development is markedly influenced by early microbial colonization of the gastrointestinal tract. As chicken are fully dependent on environmental microbes after hatching, extensive hygienic measures in production facilities are detrimental to the microbiota, resulting in low colonization resistance against pathogens. To combat enteric infections, antibiotics are frequently used, which aggravates the issue by altering gut microbiota colonization. Intervention strategies based on cultured gut bacteria are proposed to influence immune responses in chicken.

Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients.

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

SARS-CoV-2 RNA screening in routine pathology specimens.

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients' routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID-19-positive patients, (ii) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and (iii) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in seven samples from confirmed COVID-19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.