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Background Extremity replantation as well as allotransplantation aim to restore form and function of the amputated limb. Both approaches, however, are clearly limited by the ischemic time. Ex vivo perfusion (EVP), already well-established in the field of solid organ transplantation, represents a promising tool to overcome this restriction. Methods We have currently established the technical requirements to provide EVP to severed limbs in a clinical hospital setting and hereby report of a 48-hour hypothermic EVP (EVP48) of an upper extremity amputated at the level of the proximal humerus, followed by a 2-hour simulated reperfusion (2SR) with donor blood. Results Muscle biopsies revealed histopathologically well preserved, vital muscle tissue after EVP24, and partially grouped muscle fiber necrosis with predominantly vital muscle tissue after EVP48 and 2SR. Analyses of perfusate samples showed a marked decline of biochemical muscle damage markers during EVP48. Cytokine analysis disclosed an isolated increase of the proinflammatory cytokines, interleukine-6, monocyte chemotactic protein-1, and interferon-γ, during EVP and 2SR. Magnetic resonance imaging, performed after EVP48, indicated partial muscle necrosis of the intrinsic hand muscles only, while no signs for infection or inflammation were present. Conclusion Our single case experience shows the general feasibility of an amputated limb ex vivo salvage perfusion setting to allow for delayed replantation up to 24 hours. Nevertheless, an accurate prior planning is crucial to ensure successful implementation of EVP in the acute clinical setting.

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

As the central organ of the respiratory system, the human lung is responsible for supplying oxygen to the blood, which reaches the erythrocytes by diffusion through the alveolar walls and is then distributed throughout the body. By exploiting the difference in electron density detected by a phase shift in soft tissue, high-resolution X-ray phase-contrast computed tomography (XPCT) can resolve biological structures in a sub-μm range, shedding new light on the three-dimensional structure of the lungs, physiological functions and pathological mechanisms. This work presents both synchrotron and laboratory XPCT results of postmortem tissue from autopsies and biopsies embedded with various preparation protocols such as precision-cut lung slices, cryogenically fixed lung tissue, as well as paraffin and alcohol fixed tissue. The selection of pathological abnormalities includes channel of Lambert, bronchus-associated lymphoid tissue, alveolar capillary dysplasia with misalignment of pulmonary veins. Subsequently, quantification and visualization approaches are presented. The overall high image quality even of in-house XPCT scans for the case of FFPE biopsies can be exploited for a wide range of pulmonary pathologies and translated to dedicated and optimized instrumentation which could be operated in clinical setting. By using synchrotron radiation, contrast can be further increased to resolve sub-μm sized features down to the sub-cellular level. The results demonstrate that a wide range of preparation protocols including sample mounting in liquids can be used. With XPCT, poorly understood 3D structures can be identified in larger volume overview and subsequently studied in more detail at higher resolution. With the full 3D structure, the respective physiological functions of airways or vascular networks, and the different pathophysiologic mechanisms can be elucidated or at least underpinned with structural data. Moreover, synchrotron data can be used to validate laboratory protocols and provide ground truth for standardizing the method.

Idiopathic pulmonary fibrosis (IPF) is an irreversible, age-related diffuse parenchymal lung disease of poorly defined etiology. Many patients with IPF demonstrate distinctive lymphocytic interstitial infiltrations within remodeled lung tissue with uncertain pathogenetic relevance. Histopathological examination of explant lung tissue of patients with IPF revealed accentuated lymphoplasmacellular accumulations in close vicinity to, or even infiltrating, remodeled lung tissue. Similarly, we found significant accumulations of B cells interfused with T cells within remodeled lung tissue in two murine models of adenoviral TGF-β1 or bleomycin (BLM)-induced lung fibrosis. Such B cell accumulations coincided with significantly increased lung collagen deposition, lung histopathology, and worsened lung function in wild-type (WT) mice. Surprisingly, B cell–deficient µMT knockout mice exhibited similar lung tissue remodeling and worsened lung function upon either AdTGF-β1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-β1– and BLM-exposed WT mice identified a large set of commonly regulated genes, but with significant enrichment observed for Gene Ontology terms apparently not related to lung fibrogenesis. Collectively, although we observed B cell accumulations in lungs of IPF patients as well as two experimental models of lung fibrosis, comparative profiling of characteristic features of lung fibrosis between WT and B cell–deficient mice did not support a major involvement of B cells in lung fibrogenesis in mice.

Background The interest in youthful appearance and rejuvenating procedures is unbroken in our society. Besides surgical procedures, permanent fillers are utilized. The incorrect and unprofessional use of these substances, auto-injections in particular, have devastating results for patients and are challenging for the plastic surgeon. The aim of this retrospective study was to delineate the differences between permanent and non-permanent filler complications and appropriate treatment options. Methods We conducted a retrospective study and researched the hospital information system in the time period from 2001 to 2020. Patients with unprofessional use of permanent fillers, auto-injections and injections of unformulated substances were determined. Age, gender, localization, complications, length of hospital stay, comorbidities, histopathological workups and surgical salvage procedures were noted. Descriptive statistics were calculated. Results Seventeen patients were identified from 2001 till 2020. In four cases, auto-injections by the patients were the cause, whereas in the other patients the injections were performed by medical staff. Ages range from 18 to 57 years. Fourteen patients were female and three were male. The injected substances could be recognized as synthol, silicone, vaseline, fat tissue, hyaluronic acid as well as non-medical substances. Surgical procedures were necessary in eleven cases. One patient died because of the underlying diseases. Conclusion Our results indicate different sequels of filler materials injected in an unprofessional way, possible complications, conservative and surgical techniques to resolve these rare complications. We suggest a staged therapy adjusted to the clinical symptoms. Milder symptoms can be handled conservatively, whereas severe infections, skin breakdowns or persistent granuloma are justifying indications for surgical treatment. Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Pulmonary hypertension and pulmonary vascular remodeling (PVR) are common in many lung diseases leading to right ventricular dysfunction and death. Differences in PVR result in significant prognostic divergences in both the pulmonary arterial and venous compartments, as in pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD), respectively. Our goal was to identify compartment-specific molecular hallmarks of PVR, considering the risk of life-threatening pulmonary edema in PVOD, if treated by conventional pulmonary hypertension therapy. Formalin-fixed and paraffin-embedded tissues from fresh explanted human lungs of patients with PVOD (n = 19), PAH (n = 20), idiopathic pulmonary fibrosis (n = 13), and chronic obstructive pulmonary disease (n = 15), were analyzed for inflammation and kinome-related gene regulation. The generated neuronal network differentiated PVOD from PAH samples with a sensitivity of 100% and a specificity of 92% in a randomly chosen validation set, a level far superior to established diagnostic algorithms. Further, various alterations were identified regarding the gene expression of explanted lungs with PVR, compared with controls. Specifically, the dysregulation of microtubule-associated serine/threonine kinase 2 and protein-o-mannose kinase SGK196 in all disease groups suggests a key role in pulmonary vasculopathy for the first time. Our findings promise to help develop novel target-specific interventions and innovative approaches to facilitate clinical diagnostics in an elusive group of diseases.

Angiogenesis 26(2), 233 - 248 (2022). doi:10.1007/s10456-022-09860-7, A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + —macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations., Published by Springer Science + Business Media B.V, Dordrecht [u.a.]

International journal of molecular sciences 23(3), 1583 (2022). doi:10.3390/ijms23031583 special issue: "Special Issue "Viral Diseases of the Respiratory System — Molecular Mechanisms and Pathogenesis" / Special Issue Editor: Andreas Beineke, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Cells 11(4), 664 (2022). doi:10.3390/cells11040664 special issue: "Special Issue "Common Denominators of Extracellular Matrix across Tissues as Inter-Tissue Messengers and Targets for Drug Repurposing" / Special Issue Editors: Prof. Dr. Alexander Dityatev, Guest Editor; Prof. Dr. Jessica Bertrand, Guest Editor; Prof. Dr. Peter R. Mertens, Guest Editor", Published by MDPI, Basel

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies.

Interstitial lung diseases encompass a large number of entities, which are characterised by a small number of partially overlapping fibrosing injury patterns, either alone or in combination. Thus, the presently applied morphological diagnostic criteria do not reliably discriminate different interstitial lung diseases. We therefore analysed critical regulatory pathways and signalling molecules involved in pulmonary remodelling with regard to their diagnostic suitability. Using laser‐microdissection and microarray techniques, we examined the expression patterns of 45 tissue‐remodelling associated target genes in remodelled and non‐remodelled tissue samples from patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), non‐specific interstitial pneumonia (NSIP), organising pneumonia (OP) and alveolar fibroelastosis (AFE), as well as controls (81 patients in total). We found a shared usage of pivotal pathways in AFE, NSIP, OP and UIP, but also individual molecular traits, which set the fibrosing injury patterns apart from each other and correlate well with their specific morphological aspects. Comparison of the aberrant gene expression patterns demonstrated that (1) molecular profiling in fibrosing lung diseases is feasible, (2) pulmonary injury patterns can be discriminated with very high confidence on a molecular level (86–100% specificity) using individual gene subsets and (3) these findings can be adapted as suitable diagnostic adjuncts.

Pulmonary arterial hypertension (PAH) is a devastating disease with poor prognosis and limited therapeutic options. We screened for pathways that may be responsible for the abnormal phenotype of pulmonary arterial smooth muscle cells (PASMCs), a major contributor of PAH pathobiology, and identified cyclin-dependent kinases (CDKs) as overactivated kinases in specimens derived from patients with idiopathic PAH. This increased CDK activity is confirmed at the level of mRNA and protein expression in human and experimental PAH, respectively. Specific CDK inhibition by dinaciclib and palbociclib decreases PASMC proliferation via cell cycle arrest and interference with the downstream CDK-Rb (retinoblastoma protein)-E2F signaling pathway. In two experimental models of PAH (i.e., monocrotaline and Su5416/hypoxia treated rats) palbociclib reverses the elevated right ventricular systolic pressure, reduces right heart hypertrophy, restores the cardiac index, and reduces pulmonary vascular remodeling. These results demonstrate that inhibition of CDKs by palbociclib may be a therapeutic strategy in PAH., Cells of the pulmonary vasculature show a hyperproliferative phenotype in pulmonary arterial hypertension (PAH), thus contributing to the disease pathogenesis. Here the authors show that cyclin-dependent kinases are overactivated in PAH, and that their pharmacological inhibition attenuates the disease in two independent rodent models

Pulmonary veno‐occlusive disease (PVOD) is a rare lung disease characterized by fibrotic narrowing of pulmonary veins leading to pulmonary hypertension (PH) and finally to death by right heart failure. PVOD is often accompanied by pulmonary capillary hemangiomatosis (PCH), a marked abnormal proliferation of pulmonary capillaries. Both morphological patterns often occur together and are thought to be distinct manifestations of the same disease process and accordingly are classified together in group 1′ of the Nice classification of PH. The underlying mechanisms of these aberrant remodeling processes remain poorly understood. In this study, we investigated the three‐dimensional structure of these vascular lesions in the lung explant of a patient diagnosed with PVOD by μ‐computed tomography, microvascular corrosion casting, electron microscopy, immunohistochemistry, correlative light microscopy and gene expression analysis. We were able to describe multifocal intussusceptive neoangiogenesis and vascular sprouting as the three‐dimensional correlate of progressive PCH, a process dividing pre‐existing vessels by intravascular pillar formation previously only known from embryogenesis and tumor neoangiogenesis. Our findings suggest that venous occlusions in PVOD increase shear and stretching forces in the pulmonary capillary bloodstream and thereby induce intussusceptive neoangiogenesis. These findings can serve as a basis for novel approaches to the analysis of PVOD.

Adhesion barriers can be based on numerous substances. In the rat Optimized Peritoneal Adhesion Model (OPAM) the starch-based hemostats 4DryField and Arista were tested for their capability to act in a preventive manner against adhesion formation (applied as a powder that was mixed in situ with saline solution to form a barrier gel). Adhesions were scored using the established scoring systems by Lauder and Hoffmann, as well as histopathologically using the score by Zuhlke. Animals receiving saline solution were used as controls. As previously published, 4DryField reduced peritoneal adhesions significantly. However, Arista did not lead to a statistically significant reduction of adhesion formation. When comparing 4DryField and Arista applied in the same manner, only 4DryField was significantly effective in preventing peritoneal adhesions. Histopathological evaluations confirmed the results of the macroscopic investigation, leading to the conclusion that starch-based hemostats do not generally have the capability to function as effective adhesion prevention devices.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very poor prognosis and constantly growing incidence. Among other primary risks of HCC, metabolic disorders and obesity have been extensively investigated over recent decades. The latter can promote nonalcoholic fatty liver disease (NAFLD) leading to the inflammatory form of nonalcoholic steatohepatitis (NASH), that, in turn, promotes HCC. Molecular determinants of this pathogenic progression, however, remain largely undefined. In this study, we have focussed on the investigation of α-dicarbonyl compounds (α-dC), highly reactive and tightly associated with overweight-induced metabolic disorders, and studied their potential role in NAFLD and progression toward HCC using murine models. NAFLD was induced using high-fat diet (HFD). Autochthonous HCC was induced using transposon-based stable intrahepatic overexpression of oncogenic NRASG12V in mice lacking p19Arf tumor suppressor. Our study demonstrates that the HFD regimen and HCC resulted in strong upregulation of α-dC in the liver, heart, and muscles. In addition, an increase in α-dC was confirmed in sera of NAFLD and NASH patients. Furthermore, higher expression of the receptor for advanced glycation products (RAGE) was detected exclusively on immune cells and not on stroma cells in livers of mice with liver cancer progression. Our work confirms astable interplay of liver inflammation, carbonyl stress mediated by α-dC, and upregulated RAGE expression on CD8+ Tand natural killer (NK) cells in situ in NAFLD and HCC, as key factors/determinants in liver disease progression. The obtained findings underline the role of α-dC and RAGE+CD8+ Tand RAGE+ NK cells as biomarkers and candidates for a local therapeutic intervention in NAFLD and malignant liver disease., Graphical abstract

Inflammatory processes are crucial in atherosclerosis and atherothrombosis. This study aimed to identify a cytokine-pattern that is associated with plaque-vulnerability or symptomatic state in comprehensively investigated patients with symptomatic (sCS) and asymptomatic carotid stenosis (aCS). Twenty-two patients with sCS and twenty-four patients with aCS undergoing carotid endarterectomy (CEA) were considered. A cytokine-panel was measured in plasma-specimens prior to surgery and at a 90 day follow-up. Doppler-ultrasound detecting microembolic signals (MES) in the ipsilateral middle cerebral artery was performed. Carotid plaques were analysed regarding histopathological criteria of plaque-vulnerability and presence of chemokine receptor CXCR4. Correction for multiple comparisons and logistic regression analysis adjusting for vascular risk factors, grade of stenosis, antithrombotic and statin pretreatment were applied. In sCS-patients higher plasma-levels of Fractalkine (CX3CL1), IFN-α2, IL-1β, IL-2, IL-3, IL-7 were found compared to aCS-patients. CXCR4-expression on inflammatory cells was more evident in sCS- compared to aCS-plaques and was associated with vulnerability-criteria. In contrast, plasma-cytokine-levels were not related to CXCR4-expression or other vulnerability-criteria or MES. However, in both groups distinct inter-cytokine correlation patterns, which persisted at follow-up and were more pronounced in the sCS-group could be detected. In conclusion, we identified a distinct cytokine/chemokine-network in sCS-patients with elevated and closely correlated mediators of diverse functions.

Adhesion barriers can be based on numerous substances. In the rat Optimized Peritoneal Adhesion Model (OPAM) the starch-based hemostats 4DryField and Arista were tested for their capability to act in a preventive manner against adhesion formation (applied as a powder that was mixed in situ with saline solution to form a barrier gel). Adhesions were scored using the established scoring systems by Lauder and Hoffmann, as well as histopathologically using the score by Zühlke. Animals receiving saline solution were used as controls. As previously published, 4DryField reduced peritoneal adhesions significantly. However, Arista did not lead to a statistically significant reduction of adhesion formation. When comparing 4DryField and Arista applied in the same manner, only 4DryField was significantly effective in preventing peritoneal adhesions. Histopathological evaluations confirmed the results of the macroscopic investigation, leading to the conclusion that starch-based hemostats do not generally have the capability to function as effective adhesion prevention devices.

Background Unlike the vast amount of animal data available on the recellularization of allogenic decellularized heart valves (DHVs), there have only been sporadic histologic reports on such grafts in humans. Methods Two experienced cardiac pathologists independently evaluated human specimens obtained during reoperation between December 2010 and April 2017 DHVs in seven categories after automated staining (scores: 0 to 6) in comparison with published data. An optimal result of 42 points was classified as 100%. Results We found that 364 DHVs, 236 decellularized pulmonary homografts (DPHs), and 128 decellularized aortic homografts (DAHs) were implanted, and freedom from explantation was 96.1% (DAH) and 98.7% (DPH). Reoperations were because of (suspected) endocarditis in 5 of 11 patients, stenosis at the subvalvular or valvular or supravalvular level in 3 of 11 patients, planned staged reoperation in 2 of 11 patients, and 1 heart transplantation. Good reader agreement was reflected by an interagreement weighted κ of 0.783 (95% confidence interval: 0.707 to 0.859). The relative histologic score in nonendocarditis specimens was 76% ± 4.3% (maximum 81%). Intracellular procollagen type 1 production was found in recipient mesenchymal cells within the transplanted grafts. In endocarditis specimens the histologic score was significantly lower with 48% ± 7.3% (minimum 41%, p = 0.0004) because of leucocyte infiltration and matrix degradation. One DPH showed immune system-mediated graft failure. Grafts obtained during the first 12 months after implantation were not evenly repopulated with less recellularization in the inner parts; no difference was found between DAH and DPH with respect to extent of recellularization. Conclusions Substantial in vivo recellularization with noninflammatory cells was observed in this study. Spontaneous recellularization appears to require multiple months, which correspondingly has an impact on size selection for growing patients.

The pathogenetic role of angiogenesis in interstitial lung diseases (ILDs) is controversial. This study represents the first investigation of the spatial complexity and molecular motifs of microvascular architecture in important subsets of human ILD. The aim of our study was to identify specific variants of neoangiogenesis in three common pulmonary injury patterns in human ILD.We performed comprehensive and compartment-specific analysis of 24 human lung explants with usual intersitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP) and alveolar fibroelastosis (AFE) using histopathology, microvascular corrosion casting, micro-comupted tomography based volumetry and gene expression analysis using Nanostring as well as immunohistochemistry to assess remodelling-associated angiogenesis.Morphometrical assessment of vessel diameters and intervascular distances showed significant differences in neoangiogenesis in characteristically remodelled areas of UIP, NSIP and AFE lungs. Likewise, gene expression analysis revealed distinct and specific angiogenic profiles in UIP, NSIP and AFE lungs.Whereas UIP lungs showed a higher density of upstream vascularity and lower density in perifocal blood vessels, NSIP and AFE lungs revealed densely packed alveolar septal blood vessels. Vascular remodelling in NSIP and AFE is characterised by a prominent intussusceptive neoangiogenesis, in contrast to UIP, in which sprouting of new vessels into the fibrotic areas is characteristic. The molecular analyses of the gene expression provide a foundation for understanding these fundamental differences between AFE and UIP and give insight into the cellular functions involved.