Evaluation of the yeast phase-specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin sandwich ELISA to detect Talaromyces marneffei antigen in human urine.

Talaromyces (Penicillium) marneffei (TM) is an important, but neglected, thermally dimorphic fungus. It is the pathogenic cause of talaromycosis, which is strongly associated with the immunodeficiency state present in individuals with advanced HIV disease. The purpose of this study was to develop a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of T. marneffei cytoplasmic yeast antigen (TM CYA) in human urine. Monoclonal antibody (MAb) 4D1 specifically binds to TM CYA. Galanthus nivalis agglutinin (GNA), a mannose -binding lectin, recognizes and binds to mannose residues of TM CYA. For the sandwich ELISA, the microplate was coated with GNA as the capturing molecule for absorbing immune complexes of MAb 4D1-TM CYA. The MAb 4D1-GNA sandwich ELISA did not detect a cross-reaction with other antigens from other fungi or bacteria. Seventy-four urine samples from patients with blood culture -confirmed talaromycosis and 229 urine samples from people without talaromycosis residing in the endemic area were subjected to the MAb 4D1-GNA sandwich ELISA. At an optical density (OD) cutoff value of 0.356, the sensitivity was 89.19% [95% confidence interval (CI): 79.80% -95.22%]; the specificity was 98.69% (95% CI: 96.22% -99.73%). The diagnostic performance of the MAb 4D1-GNA sandwich ELISA was highly consistent with those of blood culture and the Platelia Aspergillus galactomannan (GM) ELISA kit. Collectively, the MAb 4D1-GNA sandwich ELISA is a promising technique for the rapid diagnosis of T. marneffei infection, which would facilitate the early treatment of patients with talaromycosis and it may be used to monitor treatment responses.

Expression of Cytokine Profiles in Human THP-1 Cells during Phase Transition of Talaromyces marneffei.

Talaromyces marneffei, a dimorphic fungus, exhibits temperature-dependent growth, existing in a filamentous form at 25 °C and as a yeast at 37 °C. Several studies have highlighted the important roles of macrophages in defense against T. marneffei infection. However, the immune responses to the interaction of macrophages with T. marneffei cells during phase transition require further investigation. This study reports the expression of cytokine profiles in human THP-1 cells during infection by T. marneffei. THP-1 cells were infected with T. marneffei conidia at different multiplicity of infections (MOIs). Surviving conidia transformed into yeasts after phagocytosis by macrophages, and the number of yeasts gradually increased over 36 h. The transcription and secretion levels of pro- and anti-inflammatory cytokines were examined at different times by qRT-PCR and ELISA. Transcription levels of IL-8, IL-12, IL-1ß, and TNF-α increased significantly at 12 or 24 h and then slightly decreased at 36 h. In contrast, the transcription levels of IL-6, IL-10, and TGF-ß gradually increased at all MOIs. The levels of IL-6 and IL-10 secretion corresponded to their levels of transcription. These results indicated that as the number of intracellular yeasts increased, the infected macrophages first underwent slight M1 polarization before shifting to M2 polarization. This polarization transition was confirmed by the fungicidal ability and the expression of macrophage surface markers. By inducing the M2-type polarization of macrophages, the intracellular T. marneffei cells can successfully evade the immune response. Our study provides a novel insight into the immune characterization during the transition of T. marneffei infection and could further contribute to possible diagnostic and therapeutic interventions for this infection.

The neuroprotective effect of osthole against chronic sleep deprivation (CSD)-induced memory impairment in rats.

AIM: Sleep deprivation (SD) is a frequent health problem in modern society. Osthole (Ost), a natural coumarin, has antioxidant and neuroprotective properties. This study examined the functions of Ost in chronic sleep deprivation (CSD)-induced memory deficits in rats. MAIN METHODS: The CSD rat model was constructed by applying Sleep Interruption Apparatus (SIA). The protective effect of Ost on memory ability of CSD rats was evaluated through behavioral tests. Modafinil (MOD) was a positive control for investigating the mechanisms underlying the actions of Ost. The oxidative stress changes in the cortex and hippocampus of the rats, histological changes in CA1 region in the hippocampus and the protein expressions of neural plasticity markers were measured. The hippocampal neurons were isolated from rats for evaluating the neuroprotective effects of Ost on glutamate-induced neuron injury in vitro. KEY FINDINGS: Ost administration significantly enhanced the cognitive performance of CSD rats in the open field test, object location recognition experiment, novel object recognition experiment, and Morris water maze test. Ost could effectively normalize the levels/activities of the antioxidant enzyme system in the cortex and hippocampus. Moreover, Ost administration reversed CSD-induced abnormal state of CA1 neurocytes and the down-regulated expressions of plasticity-related genes in vivo and in vitro. Additionally, Ost also notably up-regulated the expressions of Nrf2 and HO-1 previously down-regulated in CA1 neurocytes of CSD rats and in vitro. SIGNIFICANCE: Our findings showed that Ost alleviated CSD-induced cognitive deficits, and the activation of the Nrf2/HO-1 pathway might be involved in the neuroprotective action of Ost.

[Effects of Zhuang-medical thread moxibustion combined with needle-pricking on vascular oxidative stress injury in the rat].

OBJECTIVE: To observe the effect of Zhuang-medical thread moxibustion combined with needle-pricking on vascular oxidative stress injury in oxidative stress injury rats. METHODS: Eighty Wistar rats were randomly allocated to normal control, sham operation (sham), model, and combined treatment groups (n=20 in each group). The oxidative stress injury model was established by ligation of the left sciatic nerve to induce chronic constriction injury (CCI) pain stress stimulation. Zhuang-medical thread moxibustion was applied to bilateral "Zusanli" (ST 36), once a day for 3 weeks. Needle-pricking was applied to left "Yanglingquan" (GB 34) and left "Huantiao" (GB 30), once a day for 3 weeks except Sundays. Plasma 6-keto-PGF 1α, thromboxane B 2 (TXB 2), NO and ET contents were assayed by radioimmunoassay. COX-2 immunoactivity of the femoral artery was determined by immunohistochemistry, and pathological changes of the femoral artery were detected by H. E. staining. RESULTS: Compared with the control group, the levels of plasma 6-keto-PGF 1α and NO in the model group were significantly reduced (P<0.05), while those of plasma TXB 2 and ET and COX-2 expression in the femoral artery were obviously increased in the model group (P<0.01). After moxibustion plus needle-pricking treatment, CCI-induced decrease of plasma 6-keto-PGF 1α and NO contents, and increase of plasma TXB 2 and ET and COX-2 expression levels were obviously reversed (P<0.05, P<0.01). The tubal wall of the femoral artery in rats of the model group got thicker, while that of the combined treatment group was relatively thinner, suggesting an inhibition of vascular intimal hyperplasia after the treatment. CONCLUSION: Zhuang-medical thread moxibustion combined with needle-pricking of ST 36, GB 34 and GB 30 can reduce the expression of femoral artery COX-2 and regulate the balance of both plasma PGI 2/TXA 2 and plasma NO/ET in CCI-induced oxidative stress rats, which may contribute to its effect in suppressing oxidative stress-induced vascular intimal hyperplasia.