A novel method for making cell blocks with higher cellular yield.

INTRODUCTION: Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. METHODS: CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from eleven and five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. RESULTS: The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. CONCLUSION: This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.

Severe community-acquired pneumonia caused by Chlamydia psittaci genotype E/B strain circulating among geese in Lishui city, Zhejiang province, China.

Between November 2021 and January 2022, four patients of community-acquired pneumonia were admitted to the hospitals in Lishui city, Zhejiang province, China. Their main clinical manifestations were fever and dry cough as well as radiographic infiltrate, but the empiric antimicrobial therapy or traditional Chinese medicine was not effective for their illness. Clinical specimens from the patients as well as environmental and poultry specimens were collected for the determination of the causative pathogen. The ompA gene and seven housekeeping genes of Chlamydia psittaci were successfully amplified from all the patients, and the sequences of each gene were identical to one another, suggesting that they were infected by the same strain of C. psittaci. A novel strain of C. psittaci (LS strain) was isolated from the bronchoalveolar lavage fluid of patient 2 and its whole genome was obtained. Phylogenetic analyses based on the whole-genome sequences showed that the isolate is most closely related to the strain (WS/RT/E30) identified as genotype E/B. In addition, The ompA gene and four housekeeping genes of C. psittaci were also amplified from two of four faeces samples of geese at the home of patient 2, and the sequences from geese were 100% identical to those from the patients. Accordingly, these cases could be attributed to a circulating C. psittaci strain of genotype E/B in the local geese. Therefore, there is an urgent need to strengthen the regional surveillance on C. psittaci among poultry and humans for prevention and control of the outbreak of psittacosis in the city.

Curcumin Supplementation Ameliorates Bile Cholesterol Supersaturation in Hamsters by Modulating Gut Microbiota and Cholesterol Absorption.

Curcumin is a polyphenol that has been shown to have prebiotic and cholesterol-lowering properties. This study aimed to investigate the impact of curcumin on bile cholesterol supersaturation and the potential mechanistic role of intestinal microbiota and cholesterol absorption. Male hamsters (n = 8) were fed a high-fat diet (HFD) supplemented with or without curcumin for 12 weeks. Results showed that curcumin significantly decreased cholesterol levels in the serum (from 5.10 to 4.10 mmol/L) and liver (from 64.60 to 47.72 nmol/mg protein) in HFD-fed hamsters and reduced the bile cholesterol saturation index (CSI) from 1.64 to 1.08 due to the beneficial modifications in the concentration of total bile acids (BAs), phospholipids and cholesterol (p < 0.05). Gut microbiota analysis via 16S rRNA sequencing revealed that curcumin modulated gut microbiota, predominantly increasing microbiota associated with BA metabolism and short-chain fatty acid production, which subsequently up-regulated the expression of hepatic cholesterol 7-alpha hydroxylase and increased the synthesis of bile acids (p < 0.05). Furthermore, curcumin significantly down-regulated the expression of intestinal Niemann−Pick C1-like protein 1(NPC1L1) in hamsters and reduced cholesterol absorption in Caco-2 cells (p < 0.05). Our results demonstrate that dietary curcumin has the potential to prevent bile cholesterol supersaturation through modulating the gut microbiota and inhibiting intestinal cholesterol absorption.

Oxymatrine induces mitochondria dependent apoptosis in human osteosarcoma MNNG/HOS cells through inhibition of PI3K/Akt pathway.

The cytostatic drug from traditional Chinese medicinal herb has acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Oxymatrine, classified as a quinolizidine alkaloid, is a phytochemical product derived from Sophora flavescens, and has been reported to possess anticancer activities. However, the cancer growth inhibitory effects and molecular mechanisms in human osteosarcoma MNNG/HOS cell have not been well studied. In the present study, the cytotoxic effects of oxymatrine on MNNG/HOS cells were examined by MTT and bromodeoxyuridine (BrdU) incorporation assays. The percentage of apoptotic cells and the level of mitochondrial membrane potential (Δψ m) were assayed by flow cytometry. The levels of apoptosis-related proteins were measured by Western blot analysis or enzyme assay Kit. Our results showed that treatment with oxymatrine resulted in a significant inhibition of cell proliferation and DNA synthesis in a dose-dependent manner, which has been attributed to apoptosis. Furthermore, we found that oxymatrine considerably inhibited the expression of Bcl-2 whilst increasing that of Bax. This promoted mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm, as well as the activation of caspase-9 and -3. Moreover, addition of oxymatrine to MNNG/HOS cells also attenuated phosphatidylinositol 3-kinase (PI3K) /Akt signaling pathway cascade, evidenced by the dephosphorylation of P13K and Akt. Likewise, oxymatrine significantly suppressed tumor growth in female BALB/C nude mice bearing MNNG/HOS xenograft tumors. In addition, no evidence of drug-related toxicity was identified in the treated animals by comparing the body weight increase and mortality. Therefore, these findings should be useful for understanding the apoptotic cellular mechanism mediated by oxymatrine and might offer a therapeutic potential advantage for human osteosarcoma chemoprevention or chemotherapy.