[Analysis of clinicopathological characteristics of IgG4-related sialadenitis].

Objective: To analyze the clinicopathological characteristics of IgG4-related sialadenitis (IgG4-RS). Methods: A total of 40 cases diagnosed with IgG4-RS were collected from the Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine from January 2019 to December 2022. Among them, there were 26 males and 14 females. The age range was 29-77 years old [(59.4±11.8) years old], with 23 patients being older than 60 years. The lesion site, imaging manifestations, histopathological features, serological test and treatment information of patients were collected. The expression of IgG4 and IgG proteins was detected by immunohistochemistry. Results: Submandibular region swelling was the most common initial symptom of IgG4-RS (38/40, 95.0%). All the patients having serum IgG4 levels> 1.35 g/L. Serum IgG4 levels were significantly increased in patients aged>60 years (Z=-2.45, P=0.014) and those involving multiple glands (Z=-2.04, P=0.042). Thirty six cases received major salivary gland biopsy, and all the cases showed dense lymphocyte and plasma cell infiltration. Lymphoid follicle, storiform fibrosis and obliterative phlebitis were seen in 88.9% (32/36), 63.9% (23/36), 30.6% (11/36) of the cases, respectively. Twenty one cases received labial salivary gland biopsy, 66.7% (14/21) showed lymphocyte and plasma cell infiltration, 19.0% (4/21) had lymphoid follicle structures, and 33.3% (7/21) showed no obvious histological abnormalities. No signs of fibrosis or obliterative phlebitis were observed in all labial salivary gland biopsies. And 95.0% (38/40) of cases had IgG4 positive plasma cell>10/HPF, 82.5% (33/40) of cases had IgG4/IgG positive plasma cell ratio>40%. All the patients had a decrease in serum IgG4 levels after glucocorticoid treatment, but only 21.4% (6/28) of cases had reduced to normal levels (≤1.35 g/L), and there were still significant fluctuations in serum IgG4 levels thereafter. Conclusions: IgG4-RS has a predilection for middle-aged and elderly male patients, and serum IgG4 levels are significantly related to the patient's age and whether multiple glands are involved. Labial salivary gland biopsy cannot replace submandibular gland for histopathological evaluation. It is a common phenomenon that serum IgG4 levels cannot restored to normal levels after glucocorticoid treatment. This study provides certain assistance for clinical and pathological diagnosis of IgG4-RS. This study is beneficial for further understanding IgG4-RS and improving the clinical and pathological diagnosis of the disease.

[Evaluation of p16/Ki-67 double-stained immunohistochemistry in diagnosis of human papilloma virus-positive oropharyngeal squamous cell carcinoma].

Objective: To explore the diagnostic value of p16/Ki-67 double-stained immunohistochemistry in the diagnosis of human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma(opscc) and find out the optimal index to improve the accuracy of HPV detection. Methods: A total of 153 cases, from May 2014 to May 2020, diagnosed OPSCC in Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, were selected. This cohort included 130 males and 23 females, aged (58.6±10.0) years old. HPV RNA in situ hybridization was chosen as the gold standard to detect their HPV status. p16 immunohistochemistry and p16/Ki-67 double-stained immunohistochemistry were performed on all cases, and the p16/Ki-67 double positive index including 20%, 40%, and 60% were used as the thresholds to compare their sensitivity, specificity, and positive prediction value (PPV), negative prediction value (NPV) and prognosis prediction ability. Results: Among the 153 patients with OPSCC, 114 were HPV-negative and 39 were HPV-positive, and the HPV infection rate of OPSCC patients was 25.5% (39/153). Only 58.1% (36/62) of single p16 positive cases were HPV-positive, and the prognosis of patients could not be distinguished using p16 immunohistochemistry only. Using p16/Ki-67 double staining, the accuracy of HPV positive diagnosis has been improved. The HPV diagnostic ability was the highest when the p16/Ki-67 double positive index was 40% (sensitivity=86.8%, specificity=94.8%, PPV=84.6%, NPV=95.6%, area under the curve=0.897), which could distinguish the prognosis of patients (P=0.012). Conclusions: The p16/Ki-67 double-stained immunohistochemistry can improve the accuracy of HPV positive diagnosis rate and diagnosis of HPV-positive oropharyngeal cancer is the most accurate when the double-positive index is 40% as the threshold to judge HPV status and could serve as better surrogate marker for HPV detection.

Model research on repairing meniscus injury in rabbits using bone marrow mesenchymal stem cells and silk fibroin meniscus porous scaffold.

OBJECTIVE: The purpose of this study is to evaluate the histocompatibility and feasibility of structure and function of bone marrow mesenchymal stem cells (BMSCs) and silk fibroin meniscus porous scaffolds in repairing meniscus injury in rabbits. MATERIALS AND METHODS: BMSCs were cultured and identified in vitro, silk fibroin meniscus porous scaffolds were prepared and the histocompatibility of porous scaffolds was evaluated via in vitro culture. Silk fibroin-BMSCs porous scaffolds were implanted into the meniscus defect area of New Zealand white rabbits as the experimental group, the blank scaffold group without BMSCs, pure BMSCs group and blank control group were set up with 10 rabbits in each group. Joint capsules were opened for general observation at 6 weeks and 12 weeks after operation. The specimens received the HE staining, alkaline toluidine blue staining, and immunohistochemical staining of Type I and Type II collagen and S100 protein. RESULTS: In vitro HE staining and SEM observation showed that the scaffolds showed the sponge-like structure with abundant microporous structure on the surface and inside, which had a good histocompatibility with BMSCs. At 6 and 12 weeks after operation in experimental group, meniscus-like tissues were found in the meniscus defect area. HE staining showed the fibrous cartilage-like structure and obvious collagenous fiber structure around arranged in an orderly manner. Alkaline toluidine blue staining showed the cartilage-specific glycosaminoglycan (GAG); Type I and Type II collagen and S100 protein staining were strongly positive. However, the above changes were not seen in other groups. CONCLUSIONS: BMSCs and silk fibroin meniscus porous scaffolds have a better histocompatibility and feasibility of structure and function in repairing meniscus injury in rabbits.

[Prognostic significance of proteins expression by immunohistochemical method in diffuse large B cell lymphoma].

Objective: To analyze the prognostic significance of TP53, Bcl-2, Bcl-6, Myc proteins expression by immunohistochemical method (IHC) in diffuse large B cell Lymphoma (DLBCL) . Methods: Clinical and pathologic data of 223 patients with DLBCL hospitalized in Zhejiang First Hospital from March 2009 to June 2015 were retrospectively analyzed. Results: The 223 cases, a median age of 56 years old with a male predominance, had shown a 39.0% of TP53 positive expression, 38.6% of Myc, 69.1% of Bcl-2, 56.5% of Bcl-6, and 22.7% of Myc/Bcl-2 double expression. According to Hans' classification, 27.4% were GCB and 72.6% were non-GCB. With a median follow-up of 38 (2-97) months, the 3 and 5 years survival rates were 70% and 66% , respectively. By multivariate analysis, TP53 over-expression and Myc/Bcl-2 double expression were independently associated with poor outcomes. 3-year and 5-year overall survival were 59% and 57% for patients with TP53 positive, 77% and 71% for patients with TP53 negative expression. Patients with non-GCB subtype receiving chemotherapy combined with rituximab had a higher OS than those without rituximab. But rituximab did not improve the prognosis of patients with TP53 positive. Conclusion: Myc/Bcl-2 double expression and TP53 over-expression are poor prognosis for DLBCL patients. Patients with Myc/Bcl-2 double expression have shorter OS. Patients with non-GCB subtype who received chemotherapy combined with rituximab have a better OS than those without rituximab. But rituximab does not improve the prognosis of patients with TP53 positive.

Effects of silencing annexin A5 on proliferation and invasion of human cholangiocarcinoma cell line.

OBJECTIVE: We investigated the expression of annexin A5 (ANXA5) in human cholangiocarcinoma (CCA) cell line and its effect on proliferation, migration, and apoptosis of human CCA cells. MATERIALS AND METHODS: Expression of ANXA5 was detected by fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting method in 2 human CCA cell lines, QBC939 and RBE. 3 shRNA plasmids for ANXA5 silencing (ANXA5-sh1, ANXA5-sh2, ANXA5-sh3) and 1 negative control plasmid were constructed to infect QBC939 cells. The infection efficiency, expression of ANXA5, apoptosis and cell cycle of QBC939 cell were measured separately. RESULTS: The expression of ANXA5 in QBC939 cell was significantly higher than RBE cell. Expressed ANXA5 protein in the QBC939-KD cell (QBC939 cell treated by RNAi) was significantly lower than QBC939-BC (QBC939 cell) and QBC939-NC cells (QBC939 cell treated by scramble plasmid). The ratio of G0/1 phase cells and apoptosis rate increased in QBC939-KD cell. The proliferation activity and invasion ability decreased in QBC939-KD cell compared with QBC939-NC and QBC939-BC cells. CONCLUSIONS: ANXA5 play important role in the migration and apoptosis of CCA cells. Inhibiting the expression of ANXA5 significantly reduce the proliferation, migration and invasion ability of QBC939 cells, and increase the apoptosis of QBC939 cells.

[The quantitative evaluation of early radiation-induced changes in the salivary glands using MRI].

Objective: To quantitatively evaluate the early radiation injury of salivary glands in patients with nasopharyngeal carcinoma (NPC) after intensity-modulated radiotherapy (IMRT). Methods: Twenty patients with NPC between 2014 and 2015 from the Second Affiliated Hospital of Soochow University were retrospectively analyzed.All patients underwent an MRI scan before and after IMRT.The volumes, T(1)WI, T(2)WI signal intensity(SIs) and apparent diffusion coefficient (ADCs) of the parotid and submandibular glands were measured.The relative signal intensity (RSIs) of each salivary gland was calculated with cerebrospinal fluid as control.The quantitative parameters of salivary glands were compared before and after radiotherapy. Results: The volumes (cm(3)) and T(1)WI RSIs of the parotid and submandibular glands (14.88±6.00, 5.21±1.76, 2.98±1.05, 1.88±0.42, respectively) were significantly lower than those before radiotherapy (22.26±8.26, 7.76±2.45, 3.58±1.02, 2.27±0.50, respectively) (t=9.921, 4.013, 10.126, 4.202, respectively, P=0.000 for all). The T(2)WI RSIs and ADCs (×10(-3) mm(2)/s) of the parotid and submandibular glands (0.50 ± 0.08, 0.41±0.04, 1.31±0.19, 1.50±0.13, respectively) were significantly higher than those before radiotherapy (0.45±0.07, 0.33±0.05, 1.02±0.21, 1.23±0.13, respectively) (t=-4.846, -9.276, -9.957, -10.679, respectively, P=0.000 for all). The volumes of parotid and submandibular glands were correlated with ADCs (r=-0.512, P=0.000; r=-0.358, P=0.001; respectively). The volumes and ADCs of submandibular glands were correlated with T(1)WI RSIs and T(2)WI RSIs(P<0.05). Conclusion: MRI can quantitatively evaluate the early changes of salivary glands after radiotherapy of nasopharyngeal carcinoma as a noninvasive method, and has high clinical application potential.

Differential microRNA expression in signet-ring cell carcinoma compared with tubular adenocarcinoma of human gastric cancer.

Gastric cancer is a disease with a heterogeneous pathology; its pathological mechanisms remain unclear because there is a poor understanding of its etiology. In this study, we identified differentially expressed microRNAs (miRNAs) among various gastric cancer subtypes. miRNA microarray analysis and bioinformatic analysis were used to compare miRNA expression between the signet-ring cell carcinoma and tubular adenocarcinoma subtypes of gastric cancer. Thirteen dysregulated miRNAs were identified in signet-ring cell carcinoma compared with tubular adenocarcinoma: miR-30a, miR-26b, miR-381, let-7i, miR-29c, miR-543, miR-499-3p, miR-628-3p, miR-524-5p, miR-181b, miR-1914, miR-663b, and miR-676. This is the first time that miR-499-3p, miR-628-3p, miR-524-5p, and miR-1914 have been identified in gastric cancer tissues. Bioinformatic analysis using target prediction algorithms indicated that these miRNAs are directly involved in gastric cancer pathogenesis and have different pathological mechanisms in various subtypes of signet-ring cell carcinoma and tubular adenocarcinoma. The miRNA expression patterns in different gastric adenocarcinoma subtypes may help discriminate between signet-ring cell and tubular gland cancer or other gastric cancer subtypes that would otherwise be difficult to identify using routine histological and immunohistochemical analyses. These preliminary data should be verified in further prospective studies.

ß-Catenin and AKT are promising targets for combination therapy in acute myeloid leukemia.

In this study, we confirmed that combining HHT with ACR can result in synergistic cytotoxicity to AML cells in vitro and in vivo. Combining HHT and ACR simultaneously inhibited PI3K/AKT and WNT/ß-catenin signaling in AML cells. Significant increases in growth inhibition and apoptosis were induced by an AKT inhibitor when the WNT3A gene of THP-1 cells was silenced. HHT+ACR could synergistically induce the apoptosis of CD34(+)/CD38(-) primary AML cells. These results highlight ß-catenin and AKT are promising targets for combination therapy for AML.

Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.

In tissues, collagen forms the scaffold for cell attachment and migration, and it modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli. We are constructing biomimetic environments by immobilizing a collagen-derived high-affinity cell-binding peptide P-15 in three-dimensional (3-D) templates. The cell-binding peptide can be expected to transduce mechanical forces. In their physiological environment, periodontal ligament fibroblasts (PDLF) are subject to significant mechanical forces. We have examined the behavior of human PDLF in culture on particulate bovine anorganic bone mineral (ABM) coated with P-15 (ABM-P-15). Greater numbers of cells associated with ABM-P-15 compared to ABM alone. Higher levels of incorporation of radiolabeled precursors in DNA and protein were consistent with the presence of larger numbers of cells on ABM-P-15 compared to ABM cultures. Scanning electron microscopic examination showed that cultures on ABM-P-15 generated highly oriented 3-D colonies of elongated cells and formed copious amounts of fibrous as well as membranous matrix reminiscent of ligamentous structures. PDLF cultured on ABM formed sparse monolayers with little order and a meager matrix. Alizarin Red stained the matrix of particle associated cells and inter-particle cellular bridges in P-15-associated cultures, indicating mineralization. 3-D colony formation and ordering of cells along with increased mineralization suggests that the coupling of cells to the ABM matrix through P-15 may provide a biomimetic environment permissive for cell differentiation and morphogenesis. Our studies suggest that ABM-P-15 templates may be effective as endosseous grafts, and, when seeded with PDLF, these matrices may serve as tissue engineered substitutes for autologous bone grafts.

The role in cell binding of a beta-bend within the triple helical region in collagen alpha 1 (I) chain: structural and biological evidence for conformational tautomerism on fiber surface.

In its physiological solid state, type I collagen serves as a host for many types of cells. Only the molecules on fiber surface are available for interaction. In this interfacial environment, the conformation of a cell binding domain can be expected to fluctuate between the collagen fold and a distinctive non-collagen molecular marker for recognition and allosteric binding. If the cell binding domain can be localized in contiguous residues within the exposed half of a turn of the triple helix (approximately 15 residues), the need for extensive structural modification and unraveling of the triple helix is avoided. We examined the conformational preferences and biological activity of a synthetic 15-residue peptide (P-15), analogous to the sequence 766GTPGPQGIAGQRGVV780 in the alpha 1 (I) chain. Theoretical studies showed a high potential for a stable beta-bend for the central GIAG sequence. The flanking sequences showed facile transition to extended conformations. Circular dichroism of the synthetic peptide in anisotropic solvents confirmed the presence of beta-strand and beta-bend structures. P-15 inhibited fibroblast binding to collagen in a concentration dependent manner, with near maximal inhibition occurring at a concentration of 7.2 x 10(-6) M. The temporal pattern of cell attachment was altered markedly in the presence of P-15. No inhibition was seen with a peptide P-15(AI), an analogue of P-15 with the central IA residues reversed to AI or with collagen-related peptides (Pro-Pro-Gly)10, (Pro-Hyp-Gly)10, and polyproline, and with several unrelated peptides. Our studies suggest a molecular mechanism for cell binding to collagen fibers based on a conformational transition in collagen molecules on the fiber surface. Since the energy barrier between the collagen fold and beta-strand conformation is low, a local conformational change may be possible in molecules on the fiber surface because of their location in an anisotropic environment. Our observations also suggest that the sequence incorporated in P-15 may be a specific ligand for cells. Unlike other reported cell binding peptides, the residues involved in this interaction are non-polar.

Enhanced cell attachment to anorganic bone mineral in the presence of a synthetic peptide related to collagen.

We have examined the ability of a synthetic 15-residue peptide, P-15, related to a biologically active domain of type I collagen, to promote attachment of human dermal fibroblasts to anorganic bovine bone mineral (ABM) phase. The attachment of cells increased with increasing content of P-15 on the surface of ABM particles, as seen by the increased binding of radiolabeled cells, and by light microscopy and scanning electron microscopy. Incorporation of radioactive precursors of DNA and protein synthesis showed that cells on P-15-coated ABM synthesized over twofold the amount of DNA and protein than did cells on uncoated ABM. Fibroblasts attached to ABM in the presence of P-15 formed three-dimensional colonies. Cellular bridges formed between adjacent particles which aggregated in clusters with tissue-like structure. Cultures on ABM.P-15 stained for alkaline phosphatase. These observations suggest that P-15-coated ABM may be a useful matrix for bone repair.

Regulation of prolyl hydroxylase by ascorbic acid is mediated by polyADP-ribose synthesis.

While it is well known that cellular prolyl hydroxylase activity is increased in the presence of ascorbic acid, the mechanism of this modulation is not fully understood. Ascorbic acid is known to generate reactive oxygen radicals which are involved in the regulation of gene expression through mechanisms involving the synthesis of polyADP-ribose in the nucleus. We examined a possible role for this mechanism in modulating prolyl hydroxylase activity in cultures of human fetal (20 week) and neonatal (foreskin) dermal fibroblasts and IMR-90 fibroblasts. The activity of prolyl hydroxylase in these cells increased in the presence of ascorbate. Ascorbate markedly increased the levels of polyADP-ribose synthetase. The increase in prolyl hydroxylase activity was abolished or decreased by inhibitors of polyADP-ribose synthesis. Our studies suggest that ascorbate may regulate the cellular activity of prolyl hydroxylase by activating epigenetic control mechanisms involving polyADP-ribose.