Reduced chemokine C-C motif ligand 1 expression may negatively regulate colorectal cancer progression at liver metastatic sites.

Colorectal cancer (CRC) liver metastasis, albeit a stage-IV disease, is completely curable by surgical resection in selected patients. In addressing the molecular basics of this phenomenon, differentially expressed genes at primary and liver metastatic sites were screened by RNA sequencing with the use of paraffin-embedded surgical specimens. Chemokine C-C motif ligand 1 (CCL1), a chemotactic factor for a ligand of the chemokine C-C motif receptor 8 (CCR8), was isolated as one of the differentially expressed genes. Histological analysis revealed that the number of CCL1-positive cells, mainly tumour associated macrophages (TAMs) located in the stroma of CRC, decreased significantly at liver metastatic sites, while the expression level of CCR8 on CRC remained unchanged. To explore the biological significance of the CCL1-CCR8 axis in CRC, CCR8-positive CRC cell line Colo320DM was used to assess the effect of the CCL1-CCR8 axis on major signalling pathways, epithelial mesenchymal transition induction and cell motility. Upon stimulation of recombinant CCL1 (rCCL1), phosphorylation of AKT was observed in Colo320DM cells; on the other hand, the corresponding significant increase in MMP-2 levels demonstrated by RT-qPCR was nullified by siRNA (siCCR8). In the scratch test, rCCL1 treatment significantly increased the motility of Colo320DM cells, which was similarly nullified by siCCR8. Thus, the activation of the CCL1-CCR8 axis is a positive regulator of CRC tumour progression. Reduced CCL1 expression of TAMs at liver metastatic sites may partly explain the unique slow tumour progression of CRC, thus providing for a grace period for radical resection of metastatic lesions.

Technetium-99m-pyrophosphate imaging-based computed tomography-guided core-needle biopsy of internal oblique muscle in wild-type transthyretin cardiac amyloidosis.

BACKGROUND: Technetium-99m-pyrophosphate (99mTc-PYP) uptake in the internal oblique muscle (IOM), which is often observed in patients with wild-type transthyretin cardiac amyloidosis (ATTR-CA), indicates amyloid transthyretin (ATTR) deposition. OBJECTIVE: This study aimed to assess the safety and efficacy of 99mTc-PYP imaging-based computed tomography (CT)-guided core-needle biopsy of the IOM as a new extracardiac screening biopsy for confirming the presence of ATTR deposits. METHODS: Patients with suspected ATTR-CA in whom myocardial tracer uptake was detected on chest- and abdomen-centered images of 99mTc-PYP scintigraphy underwent CT-guided core-needle biopsy at the site with the highest tracer uptake in the IOM between September 2021 and November 2022. RESULTS: All 18 consecutive patients (mean age, 86.3 years ± 6.5; 61.1% male) enrolled in the study showed 99mTc-PYP uptake into the IOM. Adequate tissue samples were obtained from all patients except one without serious complications. Immunohistochemical analysis confirmed ATTR deposits in 16/18 (88.9%) patients. In the remaining two patients, ATTR deposits were observed via endomyocardial biopsy. All patients were diagnosed with wild-type ATTR-CA based on transthyretin gene sequence testing results. CONCLUSION: In wild-type ATTR-CA, 99mTc-PYP imaging-based CT-guided core-needle biopsy of the IOM could be used as an extracardiac screening biopsy to confirm the presence of ATTR deposits.

Glomangiopericytoma with CTNNB1 mutation.

Glomangiopericytoma (GPC) is a rare tumour, accounting <0.5% of all nasal cavity tumours. It is classified as borderline malignant to indolent and is associated with perivascular myeloid cells. We report a case of GPC in a woman in her 80s who presented with nasal obstruction. The tumour originated from the nasal septum and was resected via endoscopic sinus surgery. Histopathological examination revealed dense spindle-shaped to oval-shaped mesenchymal cells forming tumour nodules with staghorn-like vessel formation. Immunohistochemical analysis revealed that the tumour cells were positive for α-smooth muscle actin, CD34, ß-catenin and cyclin D1, whereas they were negative for STAT6. The percentage of Ki-67-positive cells was approximately 2%. Recent studies have demonstrated a high frequency of ß-catenin (CTNNB1) mutations in GPC. We report a case of GPC where identifying CTNNB1 mutations (c.94G>C, p.D32H) was crucial for establishing a definitive diagnosis.

Demethylation in promoter region of severely damaged hepatocytes enhances chemokine receptor CXCR4 gene expression.

The liver is known to possess remarkable regenerative potential, but persistent inflammation or severe acute injury can lead to liver fibrosis and incomplete regeneration, ultimately resulting in liver failure. Recent studies have shown that the axis of two types of CXCL12 receptors, CXCR4 and CXCR7, plays a crucial role in liver fibrosis and regeneration. The present study aimed to investigate the regulatory factors involved in CXCR4 expression in injured liver. Immunohistochemical screening of liver tissue samples collected during liver transplantation revealed a reciprocal expression pattern between CXCR4 and MeCP2. An in vitro system involving cultured cell lines and H2O2 treatment was established to study the impact of oxidative stress on signaling pathways and epigenetic alterations that affect CXCR4 mRNA expression. Operating through distinct signaling pathways, H2O2 treatment induced a dose-dependent increase in CXCR4 expression in both hepatocyte- and intrahepatic cholangiocyte-derived cells. Treatment of the cells with trichostatin and azacytidine modulated CXCR4 expression in hepatocytes by modifying the methylation status of CpG dinucleotides located in a pair of TA repeats adjacent to the TATA box of the CXCR4 gene promoter. Only MeCP2 bound to oligonucleotides representing the TATA box region when the cytosine residues within the sequence were methylated, as revealed by electrophoretic mobility shift assay (EMSA). Methylation-specific PCR analysis of microdissected samples revealed a correlation between the loss of CpG methylation and the upregulation of CXCR4 in injured hepatocytes, replicating the findings from the in vitro study. Besides the conventional MEK/ERK and NF-κB signaling pathways that activate CXCR4 in intrahepatic cholangiocytes, the unique epigenetic modifications observed in hepatocytes might also contribute to a shift in the CXCR4-CXCR7 balance towards CXCR4, leading to irreversible liver injury and fibrosis. This study highlights the importance of epigenetic modifications in regulating CXCR4 expression in liver injury and fibrosis.

Histone Modification in Histochemistry and Cytochemistry.

Keeping chromatin in a stable state is essential for genome stability, scheduled transcription, replication, DNA repair, and precise and reliable chromosome segregation and telomere maintenance during cell division. Over the past decade, research on chromatin remodeling has made great strides whereby modification of histone proteins is a key factor involved in many of the essential cellular processes. The nuclear findings of tumor cells that pathologists routinely examine are nothing but reflections of both genomic and histone alterations. Moreover, impaired histone function is known to be related to common diseases such as diabetes and atherosclerosis, and is, therefore, considered a potential therapeutic target. The present review first outlines the physiological function of histone proteins, and second, demonstrates their alterations to pathological states, emphasizing the importance of immunohistochemistry in histopathological diagnosis.

Mucinous cystadenoma of the pancreas associated with pancreatic pseudocyst.

Mucinous cystadenoma of the pancreas is considered as a premalignant lesion, and resection is recommended. The majority of pancreatic cystic lesions are pancreatic pseudocysts, so differentiation between mucinous cystadenoma and pseudocyst is frequently required. We report a rare case of mucinous cystadenoma of the pancreas coexisting with pseudocyst. A 43-year-old woman presented with abdominal pain. Imaging examinations showed a large cystic lesion in the tail of the pancreas, and distal pancreatectomy and splenectomy were performed. Pathological examination revealed that the majority of the cystic wall comprised thick collagen fibrous connective tissue, while part of the cystic wall represented a single layer of columnar, mucin-producing epithelium without atypia. Those findings suggested mucinous cystadenoma with an inflammatory pseudocyst. The mixture of mucinous cystadenoma and pseudocyst within the same cystic lesion appears to be very rare. Complete resection of the cystic lesion seems to allow an excellent prognosis.

Acute myocardial infarction caused by coronary mucormycotic embolism.

Described here is a rare cardiac complication attributed to mucormycosis in a 63-year-old woman who developed sudden cardiac arrest and pulmonary insufficiency in the course of being treated for acute monocytic leukemia (acute myelogenous leukemia, AML M5a). At autopsy, fresh thrombi were noted in the left pulmonary artery and the left atrium. Postmortem coronary angiography revealed complete occlusion of the circumflex branch of the left coronary artery, and histological examination showed a mucormycotic embolism in the corresponding portion. Multiple small mucormycotic thrombi were also noted in both coronary and pulmonary arteries with hemorrhagic infarction in the corresponding areas.

In situ sequence-specific visualization of single methylated cytosine on tissue sections using ICON probe and rolling-circle amplification.

Since epigenetic modifications differ from cell to cell, detecting the DNA methylation status of individual cells is requisite. Therefore, it is important to conduct "morphology-based epigenetics research", in which the sequence-specific DNA methylation status is observed while maintaining tissue architecture. Here we demonstrate a novel histochemical technique that efficiently shows the presence of a single methylated cytosine in a sequence-dependent manner by applying ICON (interstrand complexation with osmium for nucleic acids) probes. By optimizing the concentration and duration of potassium osmate treatment, ICON probes selectively hybridize to methylated cytosine on tissue sections. Since the elongation process by rolling-circle amplification through the padlock probe and synchronous amplification by the hyperbranching reaction at a constant temperature efficiently amplifies the reaction, it is possible to specifically detect the presence of a single methylated cytosine. Since the ICON probe is cross-linked to the nuclear or mitochondrial DNA of the target cell, subsequent elongation and multiplication reactions proceed like a tree growing in soil with its roots firmly planted, thus facilitating the demonstration of methylated cytosine in situ. Using this novel ICON-mediated histochemical method, detection of the methylation of DNA in the regulatory region of the RANK gene in cultured cells and of mitochondrial DNA in paraffin sections of mouse cerebellar tissue was achievable. This combined ICON and rolling-circle amplification method is the first that shows evidence of the presence of a single methylated cytosine in a sequence-specific manner in paraffin sections, and is foreseen as applicable to a wide range of epigenetic studies.

99mTc-Pyrophosphate Scintigraphy Can Image Tracer Uptake in Skeletal Trunk Muscles of Transthyretin Cardiac Amyloidosis.

PURPOSE: 99mTc-pyrophosphate (99mTc-PYP) uptake in the skeletal muscles is minimal in patients with transthyretin cardiac amyloidosis (ATTR-CA) when assessed qualitatively and quantitatively. We previously demonstrated moderate- to high-grade 99mTc-PYP uptake in the subcutaneous abdominal fat of some patients with ATTR-CA and showed that this abnormal finding could reflect the regional amyloid burden of this tissue. We aimed to investigate the frequency of 99mTc-PYP uptake in skeletal trunk muscles of patients with ATTR-CA. METHODS: Chest- and abdomen-centered 99mTc-PYP scintigraphy images were obtained 2 hours after IV injections of the tracer (20 mCi) in 36 patients with ATTR-CA. The frequency of 99mTc-PYP uptake in the following 11 skeletal trunk muscles was investigated: pectoralis major, deltoid, subscapularis, infraspinatus, trapezius, latissimus dorsi, erector spinae, psoas major, abdominal oblique, rectus abdominis, and the gluteus muscles. RESULTS: Ten of the 11 muscles were involved in patients with the highest number of 99mTc-PYP uptake in the skeletal trunk muscles examined, whereas no muscle was involved in a patient with the least uptake. The muscle with the highest rate of 99mTc-PYP uptake, observed in 34 of 36 patients (94.4%), was the abdominal oblique. No tracer uptake was observed in the psoas major. The frequency of radiotracer uptake in the remaining examined muscles was between those of abdominal oblique and psoas major muscles. CONCLUSIONS: Radiotracer uptake was often detectable in some skeletal trunk muscles of ATTR-CA, although the muscles of patients examined and the skeletal trunk muscles of 1 patient showed heterogeneity in the uptake of 99mTc-PYP.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

Histochemistry, Cytochemistry and Epigenetics.

Over the past few decades, many researchers have individually identified tumor-related genes, and have accumulated information on their basic research in a database. With the development of technology that can comprehensively test the expression status within a short time, oncogene panel testing has become attainable. On the other hand, changes in gene expression that do not depend on changes in base sequences, that is, epigenetics, or more comprehensively, epigenomes, are also highly involved in the development and progression of disease. Oncogene panel tests tend to focus on DNA base mutations such as point mutations, deletions, duplications, and chimera formation. Elucidation leads to correct interpretation of diseases and treatment choices, and we are in an era where integrated understanding of the genome and epigenome is indispensable. In this review, we make every effort to cover a wide range of knowledge, including data on histone protein modification, non-coding (nc)RNA and DNA methylation, and recent application trials for demonstrating epigenetic alterations in histologic and cytologic specimens. We hope this review will help marshal the knowledge accumulated by researchers involved in genomic and epigenomic studies.

Novel animal model of soft tissue tumor due to aberrant hedgehog signaling activation in pericyte lineage.

Pericytes are pluripotent cells that enclose the endothelium of small blood vessels in the whole body. These cells are thought to play a limited role in vascular development and blood pressure regulation; however, current evidence from numerous studies suggests several significant biologic aspects of pericytes in animals. One viewpoint is that pericytes are also known as potential cellular origin of multiple soft tissue tumors. Experimental evidence of the cellular origin of pericytic tumors is still insufficient, however, and their molecular pathogenesis is poorly understood. Here, we used a conditional constitutively active Smoothened allele (Rosa-SmoM2) and Cre recombinase mice to activate hedgehog (Hh) signaling, exclusively in the monocyte/macrophage and osteoclast lineage (LysMcre) or in RANK expressing cells (RANKcre) that are recognized as osteoclast precursor cells. Mice conditionally expressing SmoM2 with LysMcre displayed no significant skeletal phenotype; surprisingly, however, RANKcre; Rosa-SmoM2 mice frequently developed progressive soft tissue tumors in regions of the leg. Genetic lineage tracing analysis uncovered a new domain of RANKcre-expressing cells in the skeletal muscle interstitial cells that display markers consistent with vascular pericytes. Neoplasms arising from these cells showed increased expression of Matrix metalloproteinases (MMPs) that are molecular indicators of malignancy. Moreover, the tumors displayed strong bone invasive potency associated with osteoclastic bone resorption. Thus, these findings provide a novel insight into tumor pathology: Hh signal activated-pericytes can be a potential cellular origin of multiple soft tissue tumors.

Amyloid deposit corresponds to technetium-99m-pyrophosphate accumulation in abdominal fat of patients with transthyretin cardiac amyloidosis.

BACKGROUND: Radionuclide imaging using bone-avid tracers plays a critical role in diagnosing transthyretin cardiac amyloidosis (ATTR-CA), but technetium-99m-pyrophosphate (PYP) rarely allows the detection of extracardiac amyloid infiltration. We retrospectively investigated the frequency of PYP uptake in the subcutaneous abdominal fat of patients with ATTR-CA and its relevance to the results of fine-needle aspiration biopsy (FNAB) of this tissue. METHODS: Chest-centered images of PYP scintigraphy were obtained 2 h after the intravenous injection of the tracer (20 mCi), and the frequency of PYP uptake in the subcutaneous abdominal fat was evaluated. Amyloid deposits of fat smears taken by subcutaneous abdominal fat FNAB were assessed by Congo red staining. RESULTS: Twenty-four patients with ATTR-CA were included. Ten (41.7%) patients showed some PYP uptake in the subcutaneous abdominal fat (positive PYP group), and 14 patients did not (negative PYP group). Amyloid deposits were detected by subcutaneous abdominal fat FNAB in 7/10 patients (70.0%) of the positive PYP group versus 0/14 patients (0%) of the negative PYP group, and the difference was significant. CONCLUSIONS: In patients with ATTR-CA, abnormal PYP uptake in the subcutaneous abdominal fat could reflect the regional amyloid deposition confirmed by FNAB of this tissue.

Macrophages are requisite for angiogenesis of type H vessels during bone regeneration in mice.

Macrophages are progenitors of osteoclasts as well as regulators of bone metabolism. Macrophages mediate not only bone formation by osteoblasts under physiological conditions, but also bone regeneration after fracture. The mechanisms of macrophages regulation of bone formation and regeneration remain unclear, however. Here, we demonstrate that the liposome-encapsulated Clodronate (Clod-lip) injected mouse model with cortical bone defect induced by drill-hole injury and targeted depletion of phagocytic macrophages exhibits impaired angiogenesis of type H vessels that couple angiogenesis and osteogenesis. Moreover, we identify Tgfbi (encoding TGFBI), Plau (encoding uPA) and Tgfb1 (encoding TGF-ß1), through RNA-seq analysis, as genes of macrophage-secreted factors mediating angiogenesis and wound healing. The relevant mRNA was highly expressed in bone marrow-derived macrophages among bone cells, as determined through qRT-PCR. Finally, we disclose that treatment with uPA inhibitor or TGF-ß receptor I, receptor II inhibitor impairs bone regeneration after injury, confirming the importance of uPA and TGF-ß1 during bone regeneration. Our findings reveal a novel mechanism of bone regeneration mediated by macrophages.

RANK- NFATc1 signaling forms positive feedback loop on rank gene expression via functional NFATc1 responsive element in rank gene promoter.

Receptor Activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts, partly through the direct binding of NFATc1, to target gene promoters. We have previously cloned a 6-kb fragment containing the 5'-flanking region of the mouse RANK gene and have demonstrated the presence of binding elements of hematological transcription factors, such as MITF, PU.1 and AP-1. Here, we demonstrated the presence of the functional NFATc1 responsive element on the RANK gene promoter. Transfection of an NFATc1-expression vector increased RANK mRNA that was subsequently nullified by NFATc1 knockdown. With the use of electrophoretic mobility shift assay (EMSA), an oligonucleotide (-388/-353) showed specific protein-DNA binding that was blockshifted with an anti-NFATc1 antibody and washed out with excess amounts of the cold consensus sequence. Co-transfection studies with the use of an NFATc1-expression vector and RANK promoter-reporter constructs showed that NFATc1 increased promoter activity 2-fold in RAW264.7 cells that was again nullified as disclosed by mutagenesis studies. Taken together, these results indicate that RANK transcription is positively regulated by the RANKL signal through the direct binding of NFATc1 to its specific binding site of the RANK gene promoter, and suggest the presence of a crucial positive feedback mechanism of gene expression that promotes accelerated terminal differentiation of RANK-positive committed precursors to mature osteoclasts.

Ruptured metastatic liver tumor secondary to a thymoma: a case report.

We report a case of rupture of a synchronous metastatic liver tumor secondary to a thymoma. A 56-year-old woman was referred to our hospital with acute abdomen. Computed tomography (CT) revealed a 10 cm diameter tumor in the left lateral segment of the liver, together with ascites, which was suggestive of intra-abdominal bleeding. She was in stable condition and hemostasis was confirmed by angiography. CT also revealed a mass in the anterior mediastinum. Elective laparoscopic left lateral segmentectomy was performed to make a pathological diagnosis and for radical resection. No peritoneal dissemination was observed and the liver tumor was curatively resected. The patient subsequently underwent thymectomy. The pathological diagnoses were thymoma with the liver metastasis. Currently, at 30 months post-treatment, she has had no tumor recurrence. Rupture of a metastatic liver tumor secondary to a thymoma is a rare condition; careful preoperative management and aggressive treatment might improve the patient's prognosis.