Histology and histopathology Vol.34, nº9 (2019)

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http://hdl.handle.net/10201/179990

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Open Access
Adult-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 Mar
The 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.
Open Access
Comparison 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 Lima
Melanin 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.
Open Access
Role 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, Masashi
The 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.
Open Access
Effects 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 Maria
Although 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).
Open Access
Atelo-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.

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