Histology and histopathology Vol.34, nº9 (2019)
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- PublicationOpen AccessAdult-onset Alexander disease with a heterozygous D128N GFAP mutation: a pathological study(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Cabrera Galván, Juan José; Martínez Martin, María Soledad; Déniz García, Daniel; Araujo Ruano, Eduardo; Travieso Aja, María del MarThe various forms of Alexander disease (AD) have been linked to heterozygous point mutations in the coding region of the Human glial fibrillary acidic protein (GFAP) gene. The aim of this study was to confirm and characterise an adult variant of AD based on the presence of Rosenthal fibres, which were identified at brain autopsy. We performed histological and immunohistochemical studies and mutation screening by cycle sequencing of exons 1, 4, 6, and 8. A heterozygous D128N GFAP mutation, previously described in three other cases of adult-onset AD (AOAD), was genetically confirmed. The mutation was seemingly sporadic. Symptoms of the female, 65-year-old patient started with occasionally asymmetric motor impairment and concluded, 23 months later, with a lack of spontaneous movement in all four limbs, reduced consciousness, an acute respiratory problem, and eventually lethal exitus. The most striking characteristics were a cerebellar syndrome with subsequent clinical signs due to brainstem and spinal cord involvement. The final diagnosis was based on a complete autopsy, detection of Rosenthal fibres, GFAP, vimentin, alpha B-crystallin, ubiquitin, hsp27, neurofilament, and synaptophysin, and the identification of the corresponding GFAP gene mutation. Blood analyses were positive for ANA and rheumatoid factor. In conclusion, this work describes sporadic, rapidly advancing AOAD in a female patient and links it with other published cases with the same mutation. Reflections are provided on the influence of vasculitis and ANA in AD as well as the presence of Rosenthal fibres in the neurohypophysis.
- PublicationOpen AccessComparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Silva Almeida Vicente, Anna Luiza; Alves Bianchini, Raquel; Laus, Ana Carolina; Macedo, Graziela; Reis, Rui Manuel; Vazquez, Vinicius de LimaMelanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffinembedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.
- PublicationOpen AccessUpregulation of glucose and amino acid transporters in micropapillary carcinoma(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Nosaka, Kanae; Makishima, Karen; Sakabe, Tomohiko; Yurugi, Yohei; Wakahara, Makoto; Kubouchi, Yasuaki; Horie, Yasushi; Umekita, YoshihisaMicropapillary carcinoma (MPC), a relatively rare histologic carcinoma observed in various organs, is associated with vascular invasion, nodal metastasis, and poor prognosis. MPC is different from papillary carcinoma as it has no fibrovascular core and is thus considered essentially hypovascular. MPCs are known to upregulate glucose transporter 1 (GLUT1) via the activation of a transcription factor, hypoxiainducible factor (HIF)-1. Here we evaluated the expression of nutrient transporters in MPCs to gain a better understanding of the system used by MPCs to compensate for their intrinsic poor vascularity. We immunohistochemically evaluated 29 MPCs including breast (n=14), lung (n=8), gastrointestinal tract (n=5), and urinary tract cancers (n=2), and compared them with non-micropapillary control cancers (n=32) regarding the expression of amino acid (ASCT1, ASCT2, LAT1, and SNAT1) and glucose (GLUT1, GLUT2) transporters. Each section was scored by the staining intensity (0-3) multiplied by the occupying area (0-10), with a possible range 0-30. The average scores of the MPC and control groups were compared by Student's or Welch's t-test according to the homoscedasticity. The MPC group showed significantly higher scores for ASCT1 (p=0.007), ASCT2 (p=0.001), GLUT1 (p<0.001), and GLUT2 (p<0.001), whereas no significant scores were noted for LAT1 and SNAT1. In conclusion, MPC could be associated with the upregulation of several nutrient transporters, which may contribute to the malignant potential by supporting the survival of cancer cells.
- PublicationOpen AccessImplications on pathogenesis and risk of oral lichen planus neoplastic transformation: an ex-vivo retrospective immunohistochemical study(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Squarzanti, Diletta Francesca; Cena, Tiziana; Sorrentino, Rita; Migliario, Mario; Chiocchetti, Annalisa; Rimondini, Lia; Azzimonti, Barbara; Valente, GuidoAims. To evaluate OPN, MCM7, Ki-67, p53, Bcl-2 and 53BP1 presence, together with the abnormal adaptive CD4 and CD8 T-cell response markers expression in a series of oral lichen planus (OLP) affected patients and assess their combined contribution for a more objective disease classification. Methods and results. In this ex-vivo retrospective analysis, biopsy specimens from 28 adults with a clinical diagnosis of OLP at different progression degree (16 reticular, 2 plaque-like, 1 erosive and 9 mixed type) were collected. Sections were immunohistochemically investigated for the proinflammatory cytokine osteopontin (OPN), alpha-beta CD4 and CD8 positive T cells, DNA replication licensing factor (MCM7), proliferating cell marker (Ki-67), apoptotic and tumor antigen (p53), apoptosis modulator (Bcl-2) and cellular response regulator to double-strand breaks tumor suppressor p53-binding protein 1 expression. Statistical analysis revealed that 53BP1 is highly represented among the OLP study patients (p<0.05). Moreover, on the basis of the quantification results of the highly expressed parameters, two illness categories with different severity were evidenced. The classification hypothesis was confirmed by i) OLP lesion persistence, ii) the development of oral severe lesions in the patients belonging to high grade activity OLP group (HGA-OLPs) and iii) the ascertainment of the same evidence both in the oral squamous cell tumor controls (OSCC) and in HGA-OLP cases. Conclusion. This study completes the scenario with respect to early detection, thanks to a more precise histological analysis, for rationalizing the clinical and histological findings toward a sharable international disease scoring system.
- PublicationOpen AccessTranscription regulators are transiently expressed during the prostate gland adaptation to the hypoandrogenic environment(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Nishan, Umar; Rosa Ribeiro, Rafaela; Lenz Cesar, Carlos; Carvalho, Hernandes F.The high incidence of prostatic diseases, including malignant tumors, makes the understanding of prostate biology very important. Androgen deprivation, blockade by orchiectomy, or chemical castration causes prostate and tumor shrinkage. The gene networks involved in a cell type-specific fashion are rather unknown. This work was undertaken to identify genes with annotated function in transcription regulation that might define transitions in gene expression. A total of 15 potential regulatory genes were identified. Validation by qRT-PCR showed that Zfp703 and Arid1a exhibit expression maxima at day 1; Ash2l, Nelf, Pbx3, Eya2 at day 4; Dmrt2 at day 5 and Lbh and Sox1 at day 7 after castration. Using immunohistochemistry, we further determined that PBX3 was found in both stromal and epithelial cells, whereas ARID1A and NELF were restricted to the epithelium, and DMRT2 and EYA2 were exclusively found in the stroma. Though the proteins ZFP703 and ASH2l were not found in any experimental condition, their mRNAs were located by in situ hybridization in both epithelium and stroma. In conclusion, androgen deprivation triggers the expression of temporally regulated gene sets in both epithelial and stromal cells. These gene subsets will help establish the regulatory gene expression programs orchestrating the castration-induced remodeling of the prostate gland, and represent putative targets to increase the efficacy of androgen-deprivation to induce epithelial (and cancer) cell death.
- PublicationOpen AccessTherapeutic effects of herbal compounds in cerebral ischemia with special reference to suppression of microglia activation implicated in neurodegeneration(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Jia, Wen Ji; Yuan, Yun; Wu, Chun YunCerebral ischemia affects many especially with the ageing population. The ensuing ischemic reactions include oxidative stress, inflammation, and excitotoxicity among others. In the search for effective therapeutic strategies for cerebral ischemia, activated microglia which are the key player in neuroinflammation are now recognized as a potential therapeutic target. Microglia possess both neurotoxic and neuroprotective roles. They are protective by continuously surveilling the microenvironment, phagocytosing dead cells, secreting trophic factors and sculpting the neuronal connections by removing axons and pruning excess synapses. On the other hand, hyperactivated microglia may impair cerebral oxidative metabolism, and produce excessive proinflammatory mediators that may exacerbate the brain damage. In view of this, suppression of microglial activation has been considered a therapeutic strategy to mitigate microglia-based neuroinflammation in cerebral ischemia. However, balancing the neuroprotective and neurotoxic roles of activated microglia remains a challenging issue. Many traditional Chinese herbal agents have been used in clinic for treatment of cerebral ischemia. Here, we provide an overview of five common Chinese herbs targeting specifically microglia-mediated neuroinflammation in cerebral ischemia. It is hoped that a common parallel may be drawn from their beneficial effects especially in the latter pathological conditions for their better and effective use in the future.
- PublicationOpen AccessEffects of a probiotic on the morphology and mucin composition of pig intestine(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Desantis, Salvatore; Mastrodonato, Maria; Accogli, Gianluca; Rossi, Giacomo; Crovace, Alberto MariaAlthough the use of probiotics in human and animal medicine is growing, their mode of action remains poorly understood. This study examined the effects of a multi-strain probiotic (SLAB51™) on the morphology and carbohydrate composition of mucins secreted by goblet cells of intestinal crypts in growingfinishing pigs. Sections of duodenum, caecum and colon from pigs fed for 12 weeks with an orally administered control basal diet (No-Pro) or one with a probiotic blend (Pro) were processed for microscopic analysis and stained with (1) haematoxylin-eosin for structural and morphometrical investigation; (2) conventional histochemistry (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) for neutral, acidic nonsulphated, and sulphated mucin analysis; and (3) FITClabelled MAA-II and SNA lectins for α2,3- and α2,6- sialomucin identification. Compared with No-Pro samples, Pro samples displayed (1) increased goblet cell numbers in all investigated tract crypts; (2) an increase in acidic non-sulphomucins but a decrease in neutral, sulphated and α2,6-sialomucin-secreting goblet cells in the duodenum; (3) decreased crypt depth, an increase in α2,6-sialomucin secretory goblet cells, and a loss of goblet cell-secreting α2,3-sialomucins, which appeared on the apical surface of crypt fundus epithelial cells in the caecum; and (4) an increase in α2,6-sialomucinproducing goblet cells in the colon. Results suggest that treatment with SLAB51™ induces region-specific changes in the morphology and carbohydrate composition of mucins secreted along intestinal tracts of growing-finishing pigs. These changes could ameliorate the health status of the animals, which displayed higher growth performance and meat quality than controls (Tufarelli et al., 2017).
- PublicationOpen AccessRole of heparan sulfate 6-0 endosulfatases in intervertebral disc homeostasis(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Otsuki, Shuhei; Alvarez Garcia, Oscar; Lotz, Martin K.; Neo, MasashiThe expression of heparan sulfate endosulfatases (Sulfs) was investigated in the intervertebral disc (IVD) to clarify their role in IVD homeostasis. Sulf-1 and -2 expression were elucidated in normal and degenerated human IVD. Age-related effects on Sulf expression, type II collagen levels, and structural changes were analyzed in IVDs of wild-type (WT) and Sulf-1 knockout (Sulf-1-/- ) mice. The effect of recombinant Sulf-1 (100 ng/ml) and Sulf-1 knockdown on heparan sulfate proteoglycan and collagen expression in ATDC5 cells were examined. Finally, the effect of Sulf-1 on transforming growth factor (TGF) β1-induced signaling was evaluated. Results show that Sulf-1 and -2 levels were higher in degenerated human IVDs. In WT mice, Sulf-1 and -2 expression generally declined as the animals aged. In particular, Sulf-1 in the nucleus pulposus was higher compared with Sulf-2 at the age of 1 and 6 months and significantly declined with aging. Sulf-1-/- mice showed more severe IVD pathology than WT mice, with lower type II collagen levels in nucleus pulposus, and degeneration with type I collagen in annulus fibrosus. In vitro, Sulf-1 induced type II collagen expression and significantly increased TGF-β1- induced Smad2/3 phosphorylation in ATDC5 cells. In conclusion, Sulf-1 might play a critical role from development to maintenance of IVD homeostasis by regulating collagen expression.
- PublicationOpen AccessComparison of functional glycans between cancer stem cells and normal stem cells(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Sasaki, Norihiko; Itakura, Yoko; Gomi, Fujiya; Hirano, Kazumi; Toyoda, Masashi; Ishiwata, ToshiyukiCancer stem cells (CSCs) are a small group of cells within a tumor that preserve stemness and enhance regrowth of cancer cells. CSCs have important implications in resistance to conventional therapies and tumor relapse, although their detailed properties remain unknown. Thus, CSCs represent promising targets to improve cancer treatment. So far, a number of cell surface markers containing glycans have been exploited to identify and isolate CSCs. Cell surface glycans are well-known markers for specific cell types and also play important cellular roles, such as regulation of cell signaling. In normal stem cells, including embryonic and tissue stem cells, glycan markers in an undifferentiated state have been identified. These markers are mostly known to regulate signaling pathways required for maintenance of stemness. In contrast, CSC-specific glycans have not been well characterized yet. In this review, we summarize functional commonalities between CSCs and normal stem cells in glycan-mediated signaling pathways. Identification of CSC-specific glycans may lead to early diagnosis and radical treatment of cancer.
- PublicationOpen AccessPancreatic islet (of Langerhans) revisited(2019) Mandarim de Lacerda, Carlos A.One hundred and fifty years ago, Paul Langerhans described what would come to be known as pancreatic ‘islet of Langerhans’. Since then, we have accumulated knowledge about the pancreatic islet, the cells that exist there and the hormones secreted by these cells. The increasing prevalence of obesity, diabetes and Alzheimer’s disease in the population (three conditions that are linked to pancreatic islet function), the islet has been playing a significant role in endocrinological and metabolic studies searching how we can protect the pancreatic islet and its cell content, or how we can regenerate it. This review will be interested in the most recent and relevant aspects of knowledge regarding the pancreatic islet, always mentioning the evolution of knowledge and future perspectives for the treatment of diabetes and Alzheimer’s disease. The most recent research with microRNAs and islet culture and pseudoislet culture (organoids) allows predicting advances in knowledge with new drugs to act on the islet/cells (such as the hormone glucagon-like peptide (GLP) -1) as well as induction of other islet cells like alpha-cells and delta-cells to transform into beta-cells.
- PublicationOpen AccessAtelo-collagen type I bovine bone substitute and membrane in guided bone regeneration: a series of clinical cases and histopathological assessments(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Lucaciu, Ondine; Apostu, Dragos; Mester, Alexandru; Septimiu Campian, Radu; Gheban, Dan; Miron, Richard J.Absorbable atelo-collagen type 1 represents a new approach for guided bone regeneration with several reported advantages such as: osteoblast attachment, proliferation, mineralization potential, absorption of growth factors and inhibition of bacterial pathogen colonization. The aim of this study was to assess the clinical, radiological (preoperative width, re-entry width, gain), Periotest measurements and histologic benefits of atelo-collagen-derived bovine bone grafts (ImploBone) in combination with an atelo-collagen type I barrier membrane (ImploSorb) for guided bone regeneration (GBR) of atrophic alveolar crest in thirteen patients. Eleven patients underwent simultaneous GBR with implant insertion, two had initial GBR procedure followed by implant placement after 6 months of healing. Ridge augmentation was performed using an atelo-collagen membrane (ImploSorb, Bioimplon, Germany) and a combination of 50% ABBM (ImploBone, granule size 0.5-1mm, BioImplon Germany) mixed with 50% autologous bone. It was found that simultaneous GBR with implant placement resulted in a 35% gain at bone defect level (preoperative width 5.03±1.25 mm, re-entry width 6.81±0.98 mm, gain 1.78±1.71 mm). Implant placement performed in a 2 stage surgery 6 months following GBR was linked with a 63.9% gain at bone defect level (preoperative width 3.79±1.10 mm, re-entry width 6.22±1.41 mm, gain 2.43±1.43 mm). The total gain in both groups was 41.9% utilizing these novel biomaterials (preoperative width 4.68±1.32 mm, re-entry width 6.65±1.12 mm, gain 1.96±1.64 mm). This case series study presents a protocol where GBR can be performed either simultaneously to implant placement or delayed with this innovative biomaterial to favor bone regrowth. Future randomized controlled clinical trials are needed to further validate the bonepromoting potential of atelo-collagen-based biomaterials for bone regeneration.