Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

Cancer-associated fibroblasts in the tumor microenvironment of tongue carcinoma is a heterogeneous cell population

ObjectivesTo examine different immunophenotypes of cancer-associated fibroblasts (CAFs) in tongue squamous cell carcinoma (TSCC) and to investigate how they related to clinical outcomes.MethodsSerial sections from 54 cases of TSCC were immunohistochemically stained with α-smooth muscle actin (αSMA, CAF marker) to determine CAF density, and double-immunostained with αSMA combined with CD80 and CD86 (myeloid/monocytic-derived cell markers), Nanog (mesenchymal stem cell marker) and CD133 (hematopoietic/endothelial stem cell marker). Density of cells co-expressing these marker combinations was semi-quantitatively assessed in 5 randomly selected high power fields within the tumor area and scored as 1 – one-to-five stained cells in each field, 2 – more than 5 stained cells in each field; any finding less than score 1, was allocated a score of 0.ResultsThere were 26 CAF-poor, 16 CAF-rich and 12 CAF-intermediated cases. CD86⁺αSMA⁺ cells were the most frequent (80.4%) followed by CD80⁺αSMA⁺ (72%) and Nanog⁺αSMA⁺ cells (56%). The CD133⁺αSMA⁺ phenotype was found only in association with blood vessels. High density of αSMA⁺ CAFs was associated with disease recurrence and poor survival (p < 0.05). Increased density of CD86⁺αSMA⁺ cells was significantly associated with CAF-rich tumors and with poor survival (p < 0.05).ConclusionIn TSCC, CAFs demonstrate heterogeneous and overlapping phenotypes with the myeloid/monocytic type being the most frequent and having an impact on the clinical outcomes. Further studies are needed in order to further characterize CAF phenotypes in carcinomas of various oral sites, as this may open new frontiers for personalized medicine.     

Recent Advances in Pathology: the 2019 Annual Review Issue of The Journal of Pathology

In this Annual Review Issue of The Journal of Pathology, we present 15 invited reviews on topical aspects of pathology, ranging from the impacts of the microbiome in human disease through mechanisms of cell death and autophagy to recent advances in immunity and the uses of genomics for understanding, classifying and treating human cancers. Each of the reviews is authored by experts in their fields and our intention is to provide comprehensive updates in specific areas of pathology in which there has been considerable recent progress. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Biocompatibility of various root canal filling materials ex vivo

Aim  To evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test.
Methodology  Human gingival fibroblasts were cultured ex vivo . Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 °C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test.
Results  Eluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 ± 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease.
Conclusions  RealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications.     

Effect of phenytoin and nifedipine on collagen gene expression in human gingival fibroblasts

Phenytoin (PHT), a widely used anticonvulsant, and nifedipine (NF), an anti-anginal drug, cause clinically similar gingival overgrowths in some patients. The aim of this work was to investigate their effects on collagen and protein synthesis and cellular proliferation in normal human gingival fibroblasts in vitro. Gingival fibroblasts were cultured from biopsies taken from three healthy individuals during operations on maxillary canines and incubated with various concentrations of NF (100 and 200 ng/ml) and PHT (5 and 10 micrograms/ml) for up to 7 days. The results showed that NF and PHT have a specific effect in reducing total protein and collagen synthesis but do not influence cell proliferation in healthy gingival fibroblasts in vitro. In addition the level of mRNA for type I collagen was decreased after incubation of the cells with the drugs for 1 or 2 days. The decrease in the level of type I collagen mRNA seemed to be specific since the level of type IV collagenase mRNA used as a reference RNA did not decrease.     

Interleukin-1α produced in human gingival fibroblasts induces several activities related to the progression of periodontitis by direct contact

Previous observations suggest that interleukin-1 (IL-1) may play an important role in the progression of periodontitis. In the present study, we investigated whether a cell-associated IL-1α (CAIL-lα) produced in human gingival fibroblasts (HGF) induces biological activities related to the progression of periodontitis. HGF were treated with recombinant human IL-1β (rhIL-1β) for 12 h. After that, the cell layers of HGF were washed 3 times with fresh medium and were then fixed with 1% paraformaldehyde. The fixed cell layers of HGF were used for assays for bone resorbing activity, prostaglandin E₂ (PGE₂) production and collagenase activity. Fixed cell layers of HGF treated with rhIL-1β enhanced not only calcium release from BALB/c mouse calvaria but also PGE₂ production and collagenase activity in HGF and human periodontal ligament fibroblasts (HPLF) cultured on the fixed cell layers. These activities were neutralized by treatment with monoclonal mouse anti-human IL-1α antibody, but monoclonal mouse antihuman IL-1β antibody showed no effects on these activities. The induction of these activities by fixed cell layers of HGF required direct contact between the fixed cell layers and the calvaria, HGF, or HPLF. These results suggest that CAIL-1α produced in HGF treated with rhIL-1β induces bone resorbing activity, PGE₂ production and collagenase activity in the target cells by direct contact; CAIL-lα may play an important role in the progression of periodontitis.     

雌激素对牙周膜细胞生物学性质的影响

目的：明确雌激素对体外牙周膜成纤维细胞迁移、增殖和碱性磷酸酶的影响，以探讨雌激素对牙周组织改建的影响。方法：SD大鼠来源的牙周膜成纤维细胞经传代培养至第四代，建立体外创伤模型，分别在普通培养基和含雌激素的培养基中培养，观察测量细胞迁移情况，运用MTT、酶联检测仪测定细胞的增殖速度，对细胞的ALP进行提取和测定。结果：MTT结果硅示加入雌激素对该细胞的增殖无明显影响；在含雌激素的培养基中成纤维细胞的迁移速度加快；同时牙周膜成纤维细胞的ALP表达娃著提高。结论：雌激素能明显促进牙周膜成纤维细胞的迁移，促进其分化。     

同一个体成骨细胞和牙周韧带成纤维细胞的原代培养

目的：探索用简单，高效的方法对同一个体的成骨细胞和牙周韧带成纤维细胞进行原代培养，以便迅速建立相关的体外研究模型，为进一步研究两种细胞的相互作用奠定基础。方法：实验用酶消化法结合组织块培养法获取大鼠牙周韧带成纤维细胞，用组织块移行生长法培养成骨细胞，并对细胞培养中取材的对象和部位，酶消化时间，污染的控制等进行探讨。结果：发现所获得的细胞符合成骨细胞和成纤细胞的形态特征和生物学特性，且生长良好。结论：用酶消化法结合组织块培养法培养大鼠牙周韧带成纤维细胞，其方法可行，结果可靠。