Optical microscopic study on a novel morphological classification method of multiple diagnostic features of Sarcoptes scabiei var. hominis.

Optical microscopy is the gold standard technique used to confirm the diagnosis of scabies. Multiple diagnostic features of the pathogen Sarcoptes scabiei var. hominis (S. scabiei) can be identified under a microscope and classified into 3 categories: mites, eggs and fecal pellets. However, mite and eggshell fragments can also be observed, which have been ignored in the 2020 International Alliance for the Control of Scabies (IACS) Criteria and by most researchers. In this study, we propose a novel morphological classification method that classifies multiple diagnostic features into 5 categories and 7 subcategories. Our results revealed that 65.2% (1893 of 2896) of the positive cases were confirmed through the identification of mites, eggs or fecal pellets, whereas up to 34.6% (1003 of 2896) of the positive cases were confirmed through the identification of mite or eggshell fragments. Therefore, the important diagnostic values of mite and eggshell fragments should be emphasized. Importantly, for the first time, mite and eggshell fragments were classified into 7 subcategories, some of which are easily ignored or confused with contaminating artefacts. We believe that this novel morphological classification method will be beneficial for operator training in interpreting slides and in improving the 2020 IACS Criteria.

Mechanism of protection from insulin resistance by toll-like receptor 2 deficiency in high-fat diet fed mice: involvement in macrophage polarization.

BACKGROUND: Toll-like receptor 2 (TLR2) deficiency can increase insulin sensitivity and improves glucose tolerance. However, it is not yet fully understood about its underlying mechanism. The regulation of M1/M2 macrophage polarization has been verified to involve in insulin resistance. Here, we evaluated whether the beneficial effect of TLR2 deficiency is mediated by TLR2-associated macrophage polarization in mice fed with high-fat diet (HFD). METHODS AND RESULTS: Wild-type and TLR2 knockout (TLR2-/-) mice received HFD for two months. Following intraperitoneal glucose tolerance and insulin resistance tests, peripheral monocytes were isolated, and in vitro induced for differentiation into M1 and M2 macrophages, respectively. Macrophages polarization was evaluated using flow cytometry. The expression of macrophage polarization marker genes and cytokine production in visceral adipose tissue were measured by qRT-PCR and ELISA. Compared to wild-type mice, TLR2-/- mice showed higher glucose tolerance and insulin sensitivity, along with significantly reduced the population of M1 and increased M2 count in vitro. Additionally, TLR2-/- mice demonstrated higher expression of M2 marker iNOS mRNA and interleukin-10 level in adipose tissues. CONCLUSIONS: Our results reveal that TLR2 knockout-mediated macrophages M2 polarization is a crucial factor for preventing against diet-induced insulin resistance in mice. These findings deepen our knowledge about the mechanism underlying HFD-induced insulin resistance.

[Knockout of TLR2 gene attenuates insulin resistance and promotes M2 polarization of macrophages in mice].

Objective To study the effect of deletion of Toll-like receptor 2 (TLR2) gene on insulin resistance and polarization of macrophages in mice. Methods The wild-type (WT) and TLR2 knockout (TLR2-/-) C57BL/6 male mice, aged 28 days, were selected, with 12 mice in each group. The genotype of each mouse was identified by PCR. After mice were fed with basic diet for 3 months, the glucose tolerance test (GTT) and insulin resistance test (ITT) were performed. The mononuclear cells isolated from peripheral blood were stimulated with GM-CSF/IFN-γ and M-CSF/IL-4/IL-13, respectively, to induce differentiation to M1-like and M2-like macrophages. The CD11b, F4/80, CD11c, CD206 and early growth response 2 (EGR2) were detected by flow cytometry to determine the phenotype of macrophages. The levels of TNF-α, IL-6 and IL-10 in the culture supernatant of macrophages were detected using ELISA. Results The result of PCR identification was consistent with the genotype of mice. Compared to WT mice, TLR2-/- mice exhibited the significantly improved glycemic control at 30 min during GTT and the significantly increased insulin sensitivity at 15 minutes during ITT. The flow cytometry showed that M1 markers decreased and M2 macrophages increased in the TLR2-/- mice. ELISA showed that the levels of IL-6 and TNF-α significantly decreased in the culture supernatant of M1 macrophages, while the level of IL-10 significantly increased in the culture supernatant of M2 macrophages in TLR2-/- mice compared with WT mice. Conclusion TLR2 signal has an effect on the polarization of macrophages and makes macrophages tend to switch to M1 phenotype. A higher number of pro-inflammatory factors secreted by M1 macrophages contribute to a low-grade inflammation state in the body, which leads to a decrease in glucose tolerance and insulin sensitivity.

[Identification and bioinformatics analysis of differentially expressed microRNAs in mice liver tissues with a high fat diet-induced insulin resistance].

OBJECTIVE: To identify the expression levels of miR-1897-3p, miR-690 and miR-7a-5p in mice liver tissues with a high fat diet-induced insulin resistance using a stem-loop reverse transcriptional real-time fluorescence quantitative PCR( RT-q PCR)method, and predict microRNAs( miRNAs)-regulated target genes and their functions to investigate the relationship between insulin resistance and differentially expressed miRNAs. METHODS: The total of 30 liver tissue samples were obtained from 15 normal control mice and 15 test mice with a high fat diet-induced insulin resistance. The stemloop reverse transcriptional primers and the primers of real-time PCR were designed to establish a RT-q PCR method, and then the expression levels of miR-1897-3p, miR-690 and miR-7a-5p of 30 liver tissue samples were detected and analyzed. Using bioinformatics methods, the target genes of differentially expressed miRNAs were predicted, and then enriched gene ontology( GO), related signal pathways and target gene protein-target gene protein interactions were analyzed, respectively. RESULTS: Compared with control group, the expression levels of miR-1897-3p, miR-690 and miR-7a-5p detected by RT-q PCR in the group with a high-fat diet-induced insulin resistance were significantly decreased( P < 0. 05), which exhibited the similar pattern of down regulation to the previous microarray results. Bioinformatics analysis results showed that a total of 16 target genes were regulated by two differentially expressed miRNAs. Among of the 16 target gene protein, 8 proteins had interactions with ≥4 proteins. Rac1, Rhoa, Prkcz, Tgfbr2, Itch and Ube2d3 protein were located in the central node of the network, and they were associated or cross-linking with insulin signaling pathway. CONCLUSION: miR-1897-3p, miR-690 and miR-7a-5p in liver tissues may be involved in the physiopathologic process of insulin resistance, which may be affect the normal insulin signaling pathway cascade by regulating the expression of target genes.