Comparison of enzyme-linked immunosorbent assay with indirect immunofluorescence assay for the diagnosis of Mycoplasma pneumoniae infection.

BACKGROUND: The study aimed to compare enzyme-linked immunosorbent assay (ELISA) with indirect immunofluorescence assay (IFA) in the diagnosis of Mycoplasma pneumoniae infection. METHODS: From March 2016 to May 2017, 180 patients suspected with M. pneumoniae infection were enrolled. The SeroMP kit using ELISA and PNEUMOSLIDE kit using IFA were performed in parallel to detect the IgM antibodies against M. pneumoniae. Cohen's kappa statistics were used to assess the agreement between the ELISA and IFA assays, multivariate logistic regression analysis was used to evaluate risk factors for the discordance between the ELISA and IFA assays. RESULTS: The mean age of the enrolled subjects was 46.6 ± 21.1 years. For detection of M. pneumoniae infection, the positivities of the ELISA and IFA assays were 15.6% (95% CI: 11.0%, 21.6%) and 10.0% (95% CI: 6.4%, 15.3%), respectively. The total positivity was 19.4% (95% CI: 14.3%, 25.8%). The agreement between the ELISA and IFA assays was low (κ = 0.117, P < 0.001). Variables associated with discordant results between ELISA and IFA assays in multivariate analysis were as follows: male (OR: 0.366; 95% CI: 0.149, 0.899; P < 0.05), age (>33 years old; OR: 0.313; 95% CI: 0.129, 0.758; P < 0.05). CONCLUSION: In detection of M. pneumoniae infection, low agreement was found in IgM assays between the ELISA and IFA methods, female and younger age were significant risk factors for the discordance. A combination of ELISA and IFA tests would be recommended, in order to detect more patients suspected of M. pneumoniae infection in clinical practice.

Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy.

PTD-hFOXP3 protein acts as an immune regulator to convert human CD4(+)CD25(-) T cells to regulatory T-like cells.

Regulatory T cells (Tregs) are critical for maintaining self-tolerance and homeostasis, and have potential application in clinical disease therapy, such as autoimmune diseases and transplant rejection, but their numbers are limited. FOXP3 is a key transcription factor controlling Tregs development and function. Although transfection of CD4(+)CD25(-) lymphocytes with the FOXP3 gene can convert them to Treg-like cells, there is the risk of insertional mutagenesis and thus an alternative to genetic intervention is sought. The protein transduction domain (PTD) from the HIV transactivator of transcription is a useful tool to deliver protein to the cytoplasm and nucleus. In this study, we generated a fusion protein linking the human FOXP3 to PTD (PTD-hFOXP3), and explored its function in T cells. The results showed that the PTD rapidly and effectively delivered the hFOXP3 protein into cells where it localized not only in the cytoplasm, but also to the nucleus. PTD-hFOXP3-transduced Jurkat cells (human T lymphoma cell line) and CD4(+)CD25(-) T cells failed to proliferate and produce IL-2 and IFN-γ, but produced large amounts of the cytokines IL-4, IL-10, and TGF-ß, in response to TCR stimulation in vitro. PTD-hFOXP3-transduced CD4(+)CD25(-) T cells also expressed high levels of CTLA-4 and low levels of CD25 after stimulation. Most importantly, PTD-hFOXP3-transduced T cells inhibited the proliferation of activated CD4(+)CD25(-) T cells. Furthermore, chromatin immunoprecipitation assays demonstrated that PTD-hFOXP3 can bind with the IL-2 gene promoter and repress the expression of IL-2. These results indicate that PTD-hFOXP3 has the capability to convert conventional T cells to Treg-like cells.

Clinical Exome Sequencing Identifies a Novel Mutation of the GREB1L Gene in a Chinese Family with Renal Agenesis.

Background: Renal agenesis (RA) is one of the most severe congenital anomalies of the kidney and urinary tract; it is known to be highly genetically heterogeneous. The purpose of this study was to explore the clinical significance of genetic diagnostics in a Chinese RA family. Methods: Five members of an RA family and 100 healthy people were recruited. Clinical exome sequencing was conducted to explore the underlying genetic cause in the affected family. Results: Exome sequencing identified a novel missense mutation (c.2333T>A, p.Val778Asp) in the GREB1L gene. This GREB1L variant was not detected in controls and was predicted to be highly damaging to the physiological function of the GREB1L protein. Conclusion: We identified a novel c.2333T>A variant in the GREB1L gene that extends the mutational spectrum associated with renal agenesis.

TLR7 agonist induced repression of hepatocellular carcinoma via the TLR7-IKK-NF-κB-IL6 signaling pathway.

Toll-like receptors (TLRs) are key members of innate immunity, involved in the defense against diseases, and evidence has revealed that TLR4/5 is involved in the carcinogenesis of hepatic cancer. TLR7 belongs to the TLR family, and its roles in immune-associated hepatic diseases have been well characterized; however, the consequences of agonist targeting of TLR7 in hepatic cancer have not previously been reported. The present study aimed to investigate the effects and underlying mechanisms of Imiquimod, a TLR7 agonist, on hepatic carcinogenesis by affecting the self-renewal of hepatic cancer stem cells. To detect the effects of this TLR7 agonist on hepatic cancer cells an MTT assay, mammosphere formation assay, ALDEFLUOR™ fluorescence-based stem cell sorting was used, and the potential signaling involved in the mechanism was investigated by western blot analysis. The TLR7 agonist Imiquimod demonstrated inhibitory effects on the cell proliferation and mammosphere formation of hepatic cells and stem cells, and decreased stem cell number (P<0.01). These effects may be achieved via the TLR7/IκB kinase/nuclear factor-κB/interleukin-6 signaling pathway, with decreased levels of Snail expression. The present study demonstrated the effects and mechanisms of the TLR7 agonist on hepatic cancer occurred via suppression of the self-renewal of cancer stem cells, indicating novel potential functions of the TLR7 agonist in the treatment of HCC.

Dysregulation of GTPase IMAP family members in hepatocellular cancer.

Hepatocellular carcinoma (HCC) is one of the most life­threatening diseases in the world. Members of the GTPase of the immunity­associated protein (GIMAP) family are important in regulating apoptosis in cancer cells. However, the basic mechanism of GIMAP in HCC remains to be fully elucidated. The present study was performed to investigate the dysregulation of GIMAP family members in HCC. The techniques of polymerase chain reaction analysis, immunohistochemistry and ELISA were used to analyze the expression of GIMAP5 and GIMAP6 in HCC tissues, in matched noncancerous tissue samples, and in blood samples obtained from patients with HCC and healthy subjects. It was found that the mRNA expression levels of GIMAP5 and GIMAP6 were significantly downregulated in the HCC tumor samples, compared with the levels of expression in the matched non­tumor tissue samples. Similarly, the mRNA expression levels of GIMAP5 and GIMAP6 were also significantly downregulated in the blood samples from patients with HCC, compared with the expression levels in the blood from healthy subjects. At the protein level, it was found that the GIMAP5 and GIMAP6 proteins were expressed at lower levels in the tumor tissue samples, compared with the matched normal tissue samples, and their expression levels were also lower in the blood samples from patients with HCC, compared with the blood samples from the healthy subjects. These data, demonstrating the downregulation of the mRNA and protein expression levels of GIMAP5 and GIMAP6 in the tumor tissues and blood of patients with HCC, suggested the involvement of GIMAP5 and GIMAP6 in the pathogenesis of HCC, and indicate their possible use as diagnostic markers for HCC.