Improving the detection of Helicobacter pylori in biopsies of chronic gastritis: a comparative analysis of H&E, methylene blue, Warthin-Starry, immunohistochemistry, and quantum dots immunohistochemistry.

Objectives: The objective of the study was to compare the consistency of various staining methods, including H&E, Methylene Blue, Warthin-Starry (W-S), Immunohistochemistry (IHC) and Quantum dots immunohistochemistry (QDs-IHC), in detecting Helicobacter pylori (HP) in cases of mild, moderate and severe chronic gastritis. Methods: Biopsy samples were obtained from 225 patients with chronic gastritis at the Department of Pathology, Yichang Central People's Hospital between January 2019 and October 2019. The presence of HP was detected using H&E, Methylene Blue, W-S, IHC, and QDs-IHC. Results: The positive rates for HP detection using H&E, Methylene Blue, W-S, IHC, and QDs-IHC were 42.22%, 51.11%, 53.78%, 59.11%, and 58.67%, respectively. In cases of mild chronic gastritis, the consistency of test results between H&E, Methylene Blue, W-S, and QDs-IHC with IHC were Kappa=0.196, P=0.033, Kappa=0.706, P<0.001, Kappa=0.717, P<0.001, and Kappa=0.968, P<0.001, respectively. Similarly, in cases of moderate chronic gastritis, Kappa values between H&E, Methylene Blue, W-S, and QDs-IHC with IHC were 0.356, P<0.001, 0.655, P<0.001, 0.741, P<0.001, and 0.946, P<0.001, respectively. For cases of severe chronic gastritis, the Kappa values between the staining methods and IHC were 0.271, P=0.037, 0.421, P=0.002, 0.621, P<0.001, and 1, P< 0.001, respectively. Conclusion: The study showed that the positivity rate of IHC was significantly higher than that of H&E, Methylene Blue, and W-S in detecting HP infection in chronic gastritis cases. In terms of consistency with IHC, QDs-IHC was the most reliable staining method across all severity grades, while the agreement between H&E and IHC was poor, and that between Methylene Blue and W-S with IHC was average. Pathology departments may choose the most appropriate staining method based on their specific needs, considering the staining time, contrast, and cost of each method.

Pediatric Extraspinal Subcutaneous Sacrococcygeal Myxopapillary Ependymoma: Case Report and Minireview.

ABSTRACT: A 9-year-old girl presented with a slow-growing and painless mass for 7 months in the soft tissue of the sacrococcygeal region. Magnetic resonance imaging revealed a well-circumscribed solid mass located in the subcutaneous soft tissue of the sacrococcygeal area, but not affecting bone structures. The mass was completely removed, and the disorder was diagnosed as myxopapillary ependymoma. In addition, the MYCN gene amplification status of the tumor was evaluated. Extra-axial ependymomas are very rare tumors with a tendency to metastasis, but they are usually regarded as low-grade ependymomas. Long-time surveillance and follow-up are necessary even after complete excision. Besides, we also discuss the diagnosis of primary soft tissue myxopapillary ependymoma.

Comparison of quantum dot immunofluorescence histochemistry with conventional immunohistochemistry in detecting Helicobacter pylori infection in paraffin-embedded tissues of gastric biopsy.

Quantum dots (QDs) are a new type of fluorescent label, which has been widely used in many biological and biomedical imaging applications. In this study, we used QDs-based immunofluorescence histochemistry (QDs-IHC) and conventional immunohistochemistry (IHC) techniques to perform a retrospective analysis on paraffin-embedded tissues of gastric biopsies in 203 patients (112 of which were HP positive and 91 were negative). The ability of QDs-IHC to detect Helicobacter pylori (HP) in gastric biopsies compared to IHC technology was evaluated. In our study, both methods showed consistent HP morphology and localization. The positive detection rate of HP for QDs-IHC in formalin-fixed and paraffin-embedded (FFPE) tissue was 54.7% (111/203), and the sensitivity and specificity reached 99.11% and 100%, respectively. However the positive detection rate of HP for IHC was 53.7% (109/203), with a sensitivity and specificity of 97.32% and 100%, respectively. Weak positives (1+) were detected in 2 case of QDs-IHC with negative in IHC, and moderate positives (2+) were detected in 3 case of QDs-IHC with weak positives (1+) in IHC. The consistency test showed that the two methods showed good agreement (κ = 0.980, P = 0.014), but the sensitivity of QDs-IHC was slightly higher than that of conventional IHC. Our results show that QDs-IHC has strong sensitivity and high specificity. It is superior to conventional IHC in detecting HP infection in FFPE tissues of gastric biopsy, especially in tissues with low HP content.

SpTGase plays an important role in the hemolymph clotting in mud crab (Scylla paramamosain).

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (SpTGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). SpTGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of SpTGase. Phylogenetic analysis revealed a close relation between SpTGase to its homolog EsTGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of SpTGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus, lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant SpTGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. SpTGase was found to directly interact with another important blood coagulation component clip domain serine protease (SpcSP). Moreover, knockdown of SpTGase resulted in a decreased expression of both clotting protein precursor (SppreCP) and SpcSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest SpTGase play an important role in the hemolymph clotting in mud crab S. paramamosain.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His156, Asp226, Ser321). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.

Identification of a novel clip domain serine proteinase (Sp-cSP) and its roles in innate immune system of mud crab Scylla paramamosain.

Clip domain serine proteinases and their homologs are involved in the innate immunity of invertebrates. To identify the frontline defense molecules against pathogenic infection, we isolated a novel clip domain serine proteinase (Sp-cSP) from the hemocytes of mud crab Scylla paramamosain. The full-length 1362 bp Sp-cSP contains a 1155 bp open reading frame (ORF) encoding 384 amino acids. Multiple alignment analysis showed that the putative amino acid sequence of Sp-cSP has about 52% and 51% identity with Pt-cSP2 (AFA42360) and Pt-cSP3 (AFA42361) from Portunus trituberculatus, respectively, while the similarity with other cSP sequences was lower than 30%. However, all cSP sequences possess a conserved clip domain at the N-terminal and a Tryp-SPc domain at the C-terminal. The genomic organization of Sp-cSP consists of nine exons and eight introns, with some introns containing one or more tandem repeats. RT-PCR results indicated that Sp-cSP transcripts were predominantly expressed in the subcuticular epidermis, muscle and mid-intestine, but barely detectable in the brain and heart. Further, Sp-cSP transcripts were significantly up-regulated after challenge with lipopolysaccharides (LPS), Vibrio parahaemolyticus, polyinosinic polycytidylic acid (PolyI:C) or white spot syndrome virus (WSSV). Moreover, in vitro, the recombinant Sp-cSP revealed a strong antimicrobial activity against a Gram-positive (Staphylococcus aureus) and four Gram-negative (V. parahaemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) bacteria in a dose-dependent manner. Taken together, the acute-phase response to immune challenges and the antimicrobial activity assay indicate that Sp-cSP is a potent immune protector and plays an important role in host defense against pathogen invasion in S. paramamosain.

Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain.

Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain.