Thomas F. E. Barth

Background: Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are zoonotic diseases caused by the larval stages of Echinococcus granulosus and E. multilocularis . Prior studies have identified small particles from the outer layer of parasitic lesions termed spegs (from E. granulosus ) and spems (from E. multilocularis ), which appear to interact with the human immune system, leading to lymph node enlargement. Methods: We analyzed lymph nodes from CE ( n = 3) and AE ( n = 3) patients by immunohistochemical staining for spegs/spems and CD57. Automated image analysis revealed a significantly increased number of CD57-positive cells in Echinococcus particle-positive germinal centers compared with speg-/spem-negative germinal centers. Double immunofluorescence staining was used to characterize CD57-positive cells with markers for CD2, CD3, CD4, CD8, PD-1, Granzyme B, Perforin, and T-cell intracellular antigen 1 (TIA1). Additionally, IgG4 and IgE immunostaining was performed on liver lesions and lymph nodes containing spegs and spems and compared with unaffected controls. Results: Spegs and spems were found to accumulate in the germinal centers of the lymph nodes. Affected germinal centers showed a higher number of CD57-positive cells, which coexpressed CD2, CD3, CD4, and PD-1 but lacked cytotoxic markers (CD8, Granzyme B, Perforin, TIA1), identifying them as follicular helper T cells (Tfh). Furthermore, the speg- and spem-affected liver and lymph node sections exhibited significantly increased numbers of IgG4-, IgE-, and CD57-positive cells. Conclusions: Spegs and spems are associated with changes in the host immune response, characterized by an increased accumulation of Tfh in germinal centers. This finding is accompanied by the presence of IgE and IgG4 plasma cells in the lymph node and the perilesional area, suggesting parasite-associated immunomodulatory processes in echinococcosis. Graphical

Background: Echinococcosis is a parasitic disease, caused by the larval stage of the genus Echinococcus. Echinococcusmultilocularis and Echinococcus granulosus cause alveolar echinococcosis (AE) and cystic echinococcosis (CE) in humans. Pathologists may be confronted with the larval stage of both species. Diagnosis may be difficult due to the low incidence in daily routine. However, right diagnosis of the pathologist is still the gold standard and crucial for further treatment of the patient. Summary: Macroscopically, specimens of AE show multiple small cysts and a bread-like appearance. Instead, CE manifests as solitary or multiple grape-like cysts. In this review, we give a short algorithm for the differential diagnosis on histological grounds. Hematoxylin and eosin staining is supported by an additional periodic acid-Schiff (PAS) staining, highlighting the laminated layer (LL) as key microscopic structure. Lymphocytes and fibrosis are present at the interface with the adjoining liver tissue. The main difference lies in the morphology of the LL. In the PAS staining, this deeply violet structure is fragmented and thin in AE, while in CE, it is broad and striated. Further important characteristics are the size of the cysts and the pericystic fibrosis. The different types of CT lesions correspond to different histological features. Treatment with benzimidazoles may influence morphology. Immunohistochemistry (IHC) with the monoclonal antibodies (mAbs) EmG3 and Em2G11 shows differences in staining patterns regarding AE and CE. IHC is decisive to settle the diagnosis in unclear situations and when only small particles of E. multilocularis (SPEMS) or E. granulosus (SPEGS) are present, e.g., in lymph nodes. Key Messages: The PAS-positive LL is the microscopic hallmark for the differential diagnosis of AE/CE. For further support in unclear situations, IHC with mAb EmG3 and mAb Em2G11 is advised.

Zusammenfassung Die alveoläre und die zystische Echinokokkose sind seltene Zoonosen, verursacht durch das Larvenstadium von Echinonococcus multilocularis und Echinococcus granulosus. Aufgrund ihres tumorartigen Wachstums ist sowohl makroskopisch als auch histologisch häufig eine differentialdiagnostische Abgrenzung gegenüber Leberneoplasien erforderlich. Die überwiegend – aber nicht ausschließlich – hepatischen Läsionen des Larvenstadiums sind makroskopisch charakterisiert durch eine häufig nekrotische, mikrozystische, „brotartige“, schlecht abgegrenzte Läsion bei E. multilocularis, im Gegensatz zu gut abgrenzbaren „bandnudelartigen“, teils auch „traubenartigen“ zystischen Läsionen bei E. granulosus. Histologisch sind die Läsionen bei E. multilocularis charakterisiert durch tubuläres, infiltratives Wachstum, oft einhergehend mit ausgedehnten zentralen Nekrosen und einem Randsaum aufgebaut aus Epitheloidzellen, einer Fibrosezone und daran anschließend einem lymphoplasmazellulären Infiltrat im Übergangsbereich zu nicht befallenem Lebergewebe. Bei E. granulosus ist das nichttubulär wachsende nekrotische Areal oftmals weniger stark ausgeprägt, gefolgt von einem Randsaum, identisch zu Läsionen durch E. multilocularis, wobei die Fibrosezone deutlich breiter ist. Der Nachweis des stark PAS-positiven Lamellarkörpers ist essenziell. Dieser ist im Larvenstadium von E. multilocularis schmal und fragmentiert, bei E. granulosus deutlich breiter. Der positive immunhistologische Befund mit mAk EmG3 sichert die Diagnose der Echinokokkose. In der Differentialdiagnose ist E. multilocularis in der immunhistochemischen Färbung mit den monoklonalen Antikörpern (mAk) EmG3 und Em2G11 positiv, E. granulosus sensu lato dagegen nur mit mAk EmG3. Immunhistologisch können Fragmente des Lamellarkörpers bei E. multilocularis und E. granulosus („small particles of E. multilocularis“, SPEMs; „small particles of E. granulosus“, SPEGs) nachgewiesen werden und so die Diagnose auch in kleinen Biopsien ohne Anteile des Lamellarkörpers sichern.

Background: Alveolar echinococcosis is a rare, potentially fatal parasitosis with the main manifestation site in the liver. Diagnosis already in the initial stage of the disease is important to prevent further exacerbation and possible secondary complications by early targeted therapy. Identifying alveolar echinococcosis lesions on imaging can be difficult, and making the diagnosis can be an interdisciplinary challenge, even in a specialized center. Case presentation: In a clinically symptom-free 65-year-old white female patient with type 2 diabetes mellitus, an abdominal ultrasonography performed by a colleague in private practice revealed three small hepatic nodules as incidental findings. Further workup focused primarily on the differential diagnosis of hepatic metastatic malignancy. Therefore, a sonographically guided biopsy of the liver lesions was performed under inpatient conditions. During the control sonography routinely performed after the biopsy to exclude postinterventional hemorrhage, an examiner previously uninvolved in the case noticed the typical sonomorphology of initial alveolar echinococcosis lesions in view of the biopsied nodules. The specimens that had been collected peripherally from the target lesion under the primary assumption of metastases histopathologically showed no signs of malignancy and no other landmark findings. Follow-up staining of the biopsies with regard to the recently suspected diagnosis of alveolar echinococcosis, however, remained without a target result as well. Due to the typical sonomorphology, a further biopsy was performed. During rebiopsy, the target lesion was deliberately biopsied centrally to hit the presumed annular lamellar body localized there in alveolar echinococcosis. On the basis of the samples of the second biopsy, the diagnosis of alveolar echinococcosis in the initial stage could be confirmed histopathologically, and the patient was transferred to adequate therapy. Conclusion: One of the most important differential diagnoses of hepatic alveolar echinococcosis in the initial stage is liver metastases. Knowledge of the typical sonomorphology is essential to avoid misdiagnosis. In addition, proper localization of specimen collection within an alveolar echinococcosis initial lesion is critical to enable histopathologic diagnosis. Imaging and pathology are directly complementary, and imaging can point the way to the correct histopathologic diagnosis on the basis of lesion morphology. For this, knowledge of the specifics of alveolar echinococcosis in imaging and histomorphology is necessary to integratively combine the findings.

Background: Metabolic dysfunction–associated steatotic liver disease (MASLD) progresses to metabolic dysfunction–associated steatohepatitis (MASH) and is a major cause of liver cirrhosis. Although liver inflammation is the hallmark feature of MASH versus MASLD, the involvement of the peripheral immune cell compartments in disease progression is poorly understood, and single-cell profiles of peripheral immune cells in MASLD/MASH are not known. Methods: Patients with MASLD/MASH and healthy volunteers have been prospectively enrolled in a cross-sectional study. Patients have been histologically stratified and further characterized by liver bulk RNA sequencing (RNA-Seq). Peripheral immune cells from patients and control blood samples have been comprehensively profiled using bulk and single RNA-Seq. Results: Twenty-two patients with fibrosis stage less than F3 have been histologically stratified into patients with low, medium, and high disease activity scores (NAFLD activity score [NAS]). In contrast to fibrosis, the NAS group correlated with noninvasive imaging readouts and blood biomarkers of liver damage and inflammation (ALT, AST). The prevalence of type 2 diabetes and obesity increased with the NAS stage. Bulk RNA-seq profiling of patient liver biopsies revealed gene signatures that were positively and negatively associated with NAS. Known marker genes for liver fibrosis where upregulated on RNA level. Blood bulk RNA-seq showed only moderate differences in patients versus healthy controls. In contrast, single-cell analysis of white blood cells revealed multiple alterations of immune (sub-)populations, including an increased abundance of immature B cells and myeloid suppressor cells in patients with MASLD/MASH as compared to healthy controls. Conclusions: The study gives new insights into the pathophysiology of MASLD/MASH already manifesting relatively early in peripheral immune cell compartments. This opens new avenues for the development of new biomarker diagnostics and disease therapies. Export

Chordomas are rare malignant tumors of the bone, originating from remnants of notochordal cells. The transcription factor brachyury, encoded by TBXT, serves as a critical diagnostic marker and is essential for tumor growth. While brachyury’s role in regulating the cytoskeleton during embryogenesis and tumorigenesis is well understood, the reverse—whether cytoskeletal alterations can influence brachyury levels—remains unclear. Despite advances in understanding chordoma biology, there are currently no approved targeted therapies, underscoring the need for novel therapeutic approaches. Three chordoma cell lines were treated with cytoskeletal inhibitors, including the actin-targeting compounds Cucurbitacin B (CuB) and Latrunculin B (LatB). Morphological changes, TBXT expression, and cell viability were analyzed. The effects of CuB were examined over time and across concentrations, with cell viability assessed via apoptosis and cytotoxicity assays. Microarray gene expression profiling of ten chordoma cell lines was performed to explore CuB-mediated transcriptional changes. Rescue experiments using a TBXT open reading frame vector and co-treatments with autophagy and proteasome inhibitors were conducted to elucidate the mechanisms of brachyury depletion. Both CuB and LatB induced significant morphological changes, but only CuB caused near-complete depletion of brachyury. This effect was time- and concentration-dependent, correlating with reduced cell viability driven primarily by apoptosis. Microarray analysis revealed that CuB treatment upregulated protein refolding pathways and downregulated protein glycosylation. Notably, TBXT transcription was only slightly suppressed, indicating that brachyury depletion was largely post-transcriptional. Rescue experiments and co-treatments implicated dysregulated protein refolding and endoplasmic reticulum (ER) stress as key mechanisms underlying CuB-mediated brachyury loss. This study demonstrates that actin cytoskeleton disruption by CuB depletes brachyury in chordoma cells, primarily through dysregulated protein refolding and ER stress rather than transcriptional repression. These findings suggest that targeting actin cytoskeleton dynamics or protein unfolding pathways may provide novel therapeutic approaches for chordoma treatment.