Histology and histopathology Vol.36, nº1 (2021)
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- PublicationOpen AccessFragmentation of the Golgi complex of dopaminergic neurons in human substantia nigra: new cytopathological findings in Parkinson's disease(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Tomás, Mónica; Martínez Martínez, Narcisa; Cara Esteban, Mireia; Martínez Alonso, Emma; Martínez Menárguez, José ÁngelFragmentation of the Golgi ribbon is a common feature of Parkinson's disease and other neurodegenerative diseases. This alteration could be the consequence of the anterograde and retrograde transport imbalance, α-synuclein aggregates, and/or cytoskeleton alterations. Most information on this process has been obtained from cellular and animal experimental models, and as such, there is little information available on human tissue. If the information on human tissue was available, it may help to understand the cytopathological mechanisms of this disease. In the present study, we analyzed the morphological characteristics of the Golgi complex of dopaminergic neurons in human samples of substantia nigra of control and Parkinson's disease patients. We measured the expression levels of putative molecules involved in Golgi fragmentation, including α-synuclein, tubulin, and Golgi-associated regulatory and structural proteins. We show that, as a consequence of the disease, the Golgi complex is fragmented into small stacks without vesiculation. We found that only a limited number of regulatory proteins are altered. Rab1, a small GTPase regulating endoplasmic reticulum-to-Golgi transport, is the most dramatically affected, being highly overexpressed in the surviving neurons. We found that the SNARE protein syntaxin 5 forms extracellular aggregates resembling the amyloid plaques characteristic of Alzheimer's disease. These findings may help to understand the cytopathology of Parkinson's disease.
- PublicationOpen AccessIn Memoriam: Professor Francisco Hernández-Calvo (1931-2020)(2021) Díaz Flores, Lucio; Madrid Cuevas, Juan Francisco
- PublicationOpen AccessIdentifying specific Notch1 target proteins in lung carcinoma cells(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Hassan, Wael Abdo; Ito, TakaakiBackground. The Notch signaling pathway has different roles in many human neoplasms, being either tumor-promoting or anti-proliferative. In addition, Notch signaling in carcinogenesis can be tissue dependent. The aim of the current study is to elucidate the relation between Notch1 protein expression in lung cancer cells and the following Notch related proteins: Hes1, c-Myc, Jagged1 and Jagged2. Methods. Notch1 and its related proteins were detected in human lung cancer cell lines and in 54 surgically resected different lung carcinoma tissues. Then, we used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 small cell lung carcinoma (SCLC) cells. Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into human SCLC lines; H69. Results. The expression of Hes1, c-Myc and Jagged2 is affected by Notch1 in SCLC. Conclusion. There is a strong association between the expression of Notch1 protein and the expression of Hes1, c-Myc and Jagged2 proteins, which could aid in better understanding tumorigenesis in SCLC.
- PublicationOpen AccessAngiosarcomas: histology, immunohistochemistry and molecular insights with implications for differential diagnosis(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Machado, Isidro; Giner, Francisco; Lavernia, Javier; Cruz, Julia; Traves, Víctor; Requena, Celia; Llombart, Beatriz; López Guerrero, José Antonio; Llombart Bosch, AntonioAngiosarcomas (AS) represent a heterogenous group of tumors with variable clinical presentation. AS share an important morphologic and immunohistochemical overlap with other sarcomas, hence the differential diagnosis is challenging, especially in poorly-differentiated tumors. Although molecular studies provide significant clues, especially in the differential diagnosis with other vascular neoplasms, a thorough hematoxylin and eosin analysis remains an essential tool in AS diagnosis. In this review, we discuss pathological and molecular insights with emphasis on implications for differential diagnosis in cutaneous, breast, soft tissue and visceral AS.
- PublicationOpen AccessImmunology of tissue homeostasis, ovarian cancer growth and regression, and long lasting cancer immune prophylaxis - review of literature(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Bukovsky, AntoninData on the substantial physiological role of the immune system in the organism’s ability to manage proper differentiation and function of normal tissues (tissue homeostasis), and detailed causes of the immune system’s essential role for the in vivo stimulation of cancer growth, are severely lacking. This results in a lack of effective cancer immunotherapy without adverse events, and in the lack of long-lasting cancer immune prophylaxes, particularly in ovarian cancers. Elimination of blood auto-antibodies blocking anti-cancer T cell effectors by intermittent moderate doses of cyclophosphamide, facilitation of the immune system reactivity against alloantigens of cancer cells by two subsequent blood transfusions, and augmentation of anticancer immunity by weekly intradermal injections of bacterial toxins, caused during the subsequent treatmentfree period, lasting for two to four weeks, regression of inoperable epithelial ovarian cancers and regeneration of the tremendously metastatically altered abdominal tissues into normal healthy conditions without multivisceral cytoreductive surgery, which can result in life-threatening consequences. An otherwise untreated rectal cancer, progressing over 3 years, regressed after severe toxic dermatitis lasting over one week. This was caused by an accidental consumption of a large raw shiitake mushroom. Subsequent daily consumptions of 2 g Metformin ER and honeybee propolis ethanol extract, and weekly single larger raw shiitake mushroom, which all stimulate immune system reactivity against cancer stem cells, prevented malignant recurrence over the next 29 years without recurring dermatitis, and maintained healthy organism’s conditions. These observations indicate that regression of advanced inoperable cancers and long-lasting cancer immune prophylaxis can be reached by simple approaches
- PublicationOpen AccessAdvances in translational orthopaedic research with species-specific multipotent mesenchymal stromal cells derived from the umbilical cord(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Ramallo, Melina; Carreras Sánchez, Irene; López Fernández, Alba; Vélez, Roberto; Aguirre, Màrius; Feldman, Sara; Vives, JoaquimCompliance with current regulations for the development of innovative medicines require the testing of candidate therapies in relevant translational animal models prior to human use. This poses a great challenge when the drug is composed of cells, not only because of the living nature of the active ingredient but also due to its human origin, which can subsequently lead to a xenogeneic response in the animals. Although immunosuppression is a plausible solution, this is not suitable for large animals and may also influence the results of the study by altering mechanisms of action that are, in fact, poorly understood. For this reason, a number of procedures have been developed to isolate homologous species-specific cell types to address preclinical pharmacodynamics, pharmacokinetics and toxicology. In this work, we present and discuss advances in the methodologies for derivation of multipotent Mesenchymal Stromal Cells derived from the umbilical cord, in general, and Wharton’s jelly, in particular, from medium to large animals of interest in orthopaedics research, as well as current and potential applications in studies addressing proof of concept and preclinical regulatory aspects.
- PublicationOpen AccessH3F3A G34 mutation DNA sequencing and G34W immunohistochemistry analysis in 366 cases of giant cell tumors of bone and other bone tumors(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Gong, Lihua; Bui, Marilyn M.; Zhang, Wen; Sun, Xiaoqi; Zhang, Ming; Yi, DingH3F3A mutations and the expression of glycine 34 to tryptophan (G34W) mutants in giant cell tumors of bone (GCTBs) and other bone tumors were detected to compare H3F3A mutation types and the expression of G34W-mutant protein in order to provide a theoretical basis for using H3F3A mutations as a diagnostic and differential-diagnostic tool for GCTBs. A total of 366 bone tumor cases were investigated. The cases involved 215 men and 151 women, whose median age was 29 years (3-84). The cases included GCTB (n=180), recurrent GCTB (n=19), GCTB with lung metastasis (n=5), pediatric GCTB (n=15), primary malignant GCTB (n=5), chondroblastoma (CB, n=61), chondrosarcoma grade II (n=15), dedifferentiated chondrosarcoma (n=17), chondromyxoid fibroma (n=9), aneurysmal bone cyst (n=9), nonossifying fibroma (n=9), osteosarcoma (n=16), and undifferentiated sarcoma (n=6). Sanger DNA sequencing analysis was used to detect H3F3A mutations. Immunohistochemistry was used to assess the expression of the G34W-mutated protein in these bone tumors. DNA sequencing results revealed H3F3A mutations in 95.00% of GCTBs (171/180), including glycine 34 to tryptophan (G34W, 163/180, 90.56%), glycine 34 to leucine (G34L, 3/180, 1.67%), glycine 34 to valine (G34V, 3/180, 1.67%), and glycine 34 to arginine (G34R, 2/180, 1.11%). Recurrent GCTBs mostly had the H3F3A G34W mutation (18/19, 94.74%), and GCTBs with lung metastasis all had the H3F3A G34W mutation (5/5, 100%). Pediatric GCTBs had a mutation rate of 93.33% (14/15), including one case with G34L. Four cases of primary malignant GCTB showed the H3F3A G34W mutation (4/5, 80.00%), and the classical GCTB component and malignant component showed consistent mutation types. Immunohistochemistry showed that GCTBs harboring G34W also expressed the mutant protein in tumor cell nuclei. Furthermore, one case of GCTB and one case of recurrent GCTB showed positive G34W immunostaining results despite being negative for the genetic mutation. Other bone tumors all showed wildtype expression in both DNA sequencing and immunohistochemistry. Our large-sample DNA sequencing analysis detected four different forms of mutations in GCTBs, including three rare mutation forms. The most common mutation of H3F3A was G34W, which was in accordance with the expression of G34W in GCTBs detected by immunohistochemistry. Although DNA sequencing analysis detected rare mutation types of H3F3A, false-negative results were also present due to the small number of cells in the samples. Detection of the most common (G34W) mutant protein by immunohistochemistry was more convenient. Given the high prevalence of these driver mutations, the detection of H3F3A mutant proteins can assist in the diagnosis of GCTB and its differential diagnosis from other bone tumors.
- PublicationOpen AccessOutside-in signaling by femoral cuff injury induces a distinct vascular lesion in adipose triglyceride lipase knockout mice(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Noguchi, Hirotsugu; Yamada, Sohsuke; Hirano, Ken Ichi; Yamaguchi, Satoshi; Suzuki, Akira; Guo, Xin; Zaima, Nobuhiro; Li, Ming; Kobayashi, Kunihisa; Ikeda, Yoshihiko; Nakayama, Toshiyuki; Sasaguri, YasuyukiGenetic deficiency of adipose triglyceride lipase (ATGL), a rate-limiting enzyme for intracellular triglyceride (TG) hydrolysis, causes TG-deposit cardiomyovasculopathy (TGCV), a recently identified rare cardiovascular disorder (ORPHA code: 565612) in humans. One of the major characteristics of TGCV is a novel type of diffuse and concentric coronary atherosclerosis with ATGL-deficient smooth muscle cells (SMCs). Patients with TGCV have intractable coronary artery disease. Therefore, it is crucial to investigate the mechanisms underlying vascular lesions in ATGL deficiency using animal models. Cuff injury is an experimental procedure to induce vascular remodeling with neointimal formation with SMCs after placing a cuff around the adventitial side of the artery without direct influence on endothelium. We report the effect of cuff injury on femoral arteries of ATGLknockout (ATGL–/–) mice. Cuff-induced concentric neointimal formation with migrating SMCs was exacerbated in ATGL–/– mice, mimicking atherosclerotic lesions in patients with TGCV. In the media, cell death of SMCs and loss of elastic fibers increased. Perivascular infiltrating cells expressing tumor necrosis factor-α (TNF-α) were more prominent in ATGL–/– mice than in wild-type (WT) mice. In Boyden chamber experiments, a greater number of ATGL–/– SMCs migrated in response to TNF-α compared to WT SMCs. These data, for the first time, demonstrated that outsidein signaling by cuff-induced neointimal formation where paracrine stimuli from adventitial infiltrating cells may lead to neointimal formation and mediolysis in ATGLdeficient conditions. Cuff injury might be a valuable model for understanding the mechanisms underlying the development of atherosclerotic lesions in patients with TGCV
- PublicationOpen AccessNovel PD-L1 mAb HC16 reveals upregulation of PD-L1 in BAC subtype(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Su, Bor Chyuan; Ting, Chen Hung; Lee, Kang Yun; Wu, Sheng Ming; Feng, Po Hao; Chan, Yao Fei; Chen, Jyh YihProgrammed death-ligand 1 (PD-L1) is an inhibitory transmembrane protein that can prevent autoimmune response. Upregulated PD-L1 serves as a predictive biomarker for patients who may respond well to immune checkpoint therapies. However, variable associations of PD-L1 level with prognoses have been reported. In this study, a short peptide sequence corresponding to PD-L1 amino acids 172-187 (from the extracellular Ig-like C-type domain, and with high predicted antigenicity and hydrophilicity) was used to generate a monoclonal antibody (mAb). The resultant PD-L1 mAb, clone HC16, was examined for binding specificity and reactivity in cancer cell-lines, as assessed by immunocytochemical, immunoblotting, and coimmunoprecipitation. The potential diagnostic and clinical applicability of clone HC16 was further tested using malignant tissue arrays derived from various cancer types analyzed with an automated immunohistochemical (IHC) staining platform. Additionally, tumor samples from patients diagnosed with non-small cell lung cancer (NSCLC) were analyzed by western blotting. Clone HC16 showed obvious staining activity in lung and breast cancer tissues. Interestingly, we observed that PD-L1 level was negatively associated with clinical stage in NSCLC. Strong PD-L1 expression tended to be found in patients diagnosed with bronchioloalveolar carcinoma (BAC). These results demonstrate that clone HC16 harbors good target specificity and is suitable for further development in diagnostic tools to assess PD-L1 expression in human tissues. In addition, our findings also suggest a role for PD-L1 in a non-invasive subtype of lung cancer.
- PublicationOpen AccessThe BRD4 inhibitor JQ1 protects against chronic obstructive pulmonary disease in mice by suppressing NF-κB activation(Universidad de Murcia. Departamento de Biología Celular e Histología, 2021) Liu, Yan; Huang, Zhi Zhen; Min, Li; Li, Zhi Feng; Chen, KuiObjective. To examine the effect of the BRD4 inhibitor JQ1 on mice with chronic obstructive pulmonary disease (COPD) via NF-κB. Methods. COPD models constructed by exposure to cigarette smoke and intratracheal instillation of lipopolysaccharides (LPS) in mice were treated with JQ1 (15, 25 or 50 mg/kg). HE staining was performed to observe histopathological changes in the lung tissues. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of IL-10, IFN-γ, IL-17, IL1β, IL-6, TNF-α, MMP-2, MMP-9, MDA, SOD, T-AOC and HO-1, and gelatin zymography assays were used to examine MMP-2 and MMP-9 activity. A TransAMTM NF-κB p65 detection kit was used to test NF-κB p65/DNA binding activity. Western blotting was conducted to analyze NF-κB p65 in the nucleus and its acetylation. Results. JQ1 dose-dependently improved the histopathological changes in the lung tissues and decreased the mean linear intercept (MLI), destructive index and inflammatory score of the mice with COPD. The mice with COPD showed increased levels of MMP2, MMP-9, IFN-γ, IL-17, IL-1β, IL-6 and TNF-α with decreased IL-10 level; these changes were reversed by JQ1 in a dose-dependent manner. In addition, JQ1 reduced the MDA level and increased the SOD, HO-1 and T-AOC levels in mice with COPD, with suppression of NF-κB p65 expression in the nucleus, NF-κB/p65 (Lys310) acetylation and NF-κB p65/DNA binding activity in the lung tissues. Conclusion. The BRD4 inhibitor JQ1 can downregulate MMP-2 and MMP-9 expression, reduce inflammatory responses, and alleviate oxidative stress in mice with COPD, and this mechanism might be related to the inhibition of NF-κB.