Reactive oxygen species mediated T lymphocyte abnormalities in an iron-overloaded mouse model and iron-overloaded patients with myelodysplastic syndromes.

The adverse effects of iron overload have raised more concerns as a growing number of studies reported its association with immune disorders. This study aimed to investigate alterations in the immune system by iron overload in patients with myelodysplastic syndrome (MDS) and an iron-overloaded mouse model. The peripheral blood from patients was harvested to test the effect of iron overload on the subsets of T lymphocytes, and the level of reactive oxygen species (ROS) was also evaluated. The data showed that iron-overloaded patients had a lower percentage of CD3+ T cells and disrupted T cell subsets, concomitant with higher ROS level in lymphocytes. In order to explore the mechanism, male C57Bl/6 mice were intraperitoneally injected with iron dextran at a dose of 250 mg/kg every 3 days for 4 weeks to establish an iron-overloaded mouse model and the blood of each mouse was collected for the analysis of the T lymphocyte subsets and T cell apoptosis. The results showed that iron overload could reduce the percentage of CD3+ T cells and the ratio of Th1/Th2 and Tc1/Tc2 but increase the percentage of regulatory T (Treg) cells and the ratio of CD4/CD8. We also found that iron overload induced the apoptosis of T lymphocytes and increased its ROS level. Furthermore, these effects could be partially recovered after treating with antioxidant N-acetyl-L-cysteine (NAC) or iron chelator deferasirox (DFX). Taken together, these observations indicated that iron overload could selectively affect peripheral T lymphocytes and induce an impaired cellular immunity by increasing ROS level.

Gastrodin Reduces the Severity of Status Epilepticus in the Rat Pilocarpine Model of Temporal Lobe Epilepsy by Inhibiting Nav1.6 Sodium Currents.

Temporal lobe epilepsy (TLE) is one of the most refractory types of adult epilepsy, and treatment options remain unsatisfactory. Gastrodin (GAS), a phenolic glucoside used in Chinese herbal medicine and derived from Gastrodia elata Blume, has been shown to have remarkable anticonvulsant effects on various models of epilepsy in vivo. However, the mechanisms of GAS as an anticonvulsant drug remain to be established. By utilizing a combination of behavioral surveys, immunofluorescence and electrophysiological recordings, the present study characterized the anticonvulsant effect of GAS in a pilocarpine-induced status epilepticus (SE) rat model of TLE and explored the underlying cellular mechanisms. We found that GAS pretreatment effectively reduced the severity of SE in the acute phase of TLE. Moreover, GAS protected medial entorhinal cortex (mEC) layer III neurons from neuronal death and terminated the SE-induced bursting discharge of mEC layer II neurons from SE-experienced rats. Furthermore, the current study revealed that GAS prevented the pilocarpine-induced enhancement of Nav1.6 currents (persistent (INaP) and resurgent (INaR) currents), which were reported to play a critical role in the generation of bursting spikes. Consistent with this result, GAS treatment reversed the expression of Nav1.6 protein in SE-experienced EC neurons. These results suggest that the inhibition of Nav1.6 sodium currents may be the underlying mechanism of GAS's anticonvulsant properties.

Telomerase activity increased and telomere length shortened in peripheral blood cells from patients with immune thrombocytopenia.

OBJECTIVES: To evaluate the telomere/telomerase system and its clinical significance in immune thrombocytopenia (ITP) patients. METHODS: A total of 237 ITP patients, 20 SLE patients and 200 age-and sex-matched healthy controls were included in this study. CD4(+), CD8(+) and CD19(+) lymphocytes were purified by magnetic beads sorting from peripheral blood of 37 active chronic ITP patients and 22 age-and sex-matched healthy controls. Telomerase activity was assayed by Telo TTAGGG Telomerase PCR ELISA KIT. The relative telomere length of peripheral blood mononuclear cell (PBMC) was measured by a quantitative polymerase chain reaction-based method (Q-PCR) from 200 ITP patients and 178 age-and sex-matched healthy controls. RESULTS: Telomerase activity was increased in CD4(+), CD8(+) and CD19(+) lymphocytes from ITP patients compared to those from healthy controls (p = 0.000). The level of telomerase activity in CD19(+) lymphocyte was higher than those in CD4(+) and CD8(+) lymphocytes. Telomerase activity of CD19+ lymphocytes had a modest negative correlation with platelet count in ITP patients (p = 0.042). The relative telomere length of PBMC in ITP patients was significantly shorter than that in the healthy controls (p = 0.002). Telomere length of PBMC in active ITP patients was significantly shorter than that in the controls (p = 0.000) and a tendency to be shorter even in inactive ITP patients (p = 0.065). Moreover, the telomere length in refractory and non-refractory ITP patients were both significantly shorter than that in the controls (p = 0.025; p = 0.000). However no significant difference in telomere length of PBMC was found between refractory ITP patients and non-refractory ITP patients (p = 0.234). CONCLUSION: An abnormal regulating telomere/telomerase system might be involved in the pathogenesis of ITP. Further studies may elucidate whether the telomere length could be considered as a predictive biomarker for the prognosis of ITP.

Simulation-Based Learning Combined with Case and Problem-Based Learning in the Clinical Education of Joint Surgery.

OBJECTIVE: Traditional education of clinical training mainly relies on a single mode of lecture-based learning (LBL), in which the teacher lectures and the students listen, and the teaching effect is often unsatisfactory. This study aims to explore the effect of simulation-based learning (SBL) combined with case and problem-based learning (CPBL) teaching mode in the clinical education of joint surgery. DESIGN: Through objective evaluation of joint surgery students' theoretical knowledge and clinical skills, and subjective evaluation of teaching quality by anonymous questionnaire, the teaching effects of LBL teaching mode, CPBL teaching mode and SBL combined with CPBL teaching mode in clinical teaching of joint surgery were compared. SETTING AND PARTICIPANTS: Sixty students who participated in the standardized training of residents in the Center for Joint Surgery, Southwest Hospital, Army University, China from March 2020 to September 2021 were selected and randomly divided into groups A, B, and C, with 20 students in each group. Group A adopted traditional LBL mode, group B adopted CPBL mode, and group C adopted SBL combined with CPBL mode. RESULTS: The scores of theoretical knowledge, clinical skills and total score of group C were (86.40 ± 9.76), (92.15 ± 4.49), (88.70 ± 5.75) points respectively, which were significantly higher than (78.80 ± 10.50), (86.60 ± 8.79), (81.92 ± 6.97) points in group B, and (80.50 ± 6.64), (85.35 ± 7.99), (82.44 ± 5.97) points in group A, the difference was statistically significant (p < 0.05). The scores of 5 self-evaluation items, i.e., learning interest, self-learning ability, problem-solving ability, clinical skills and comprehensive competency were (18.90 ± 1.22), (18.85 ± 1.01), (18.75 ± 1.13), (18.90 ± 1.22), (18.50 ± 1.02), (18.80 ± 0.81) points in group C, which were higher than (15.90 ± 1.41), (14.30 ± 2.47), (13.95 ± 2.01), (14.50 ± 1.63), (14.70 ± 1.38) points in group B, and (11.65 ± 2.90), (10.05 ± 1.69), (9.75 ± 1.67), (14.35 ± 1.90), (12.75 ± 2.12) points in group A, the difference was statistically significant (p <0.05). The satisfaction of students in group C (95.00%) was significantly better than that in group B (80.00%) and group A (65.00%), and the difference was statistically significant (p < 0.05). CONCLUSION: SBL combined with CPBL teaching mode can effectively improve the theoretical knowledge and clinical skills of the students, which could improve self-assessment and teaching satisfaction rate, and is worthy of application and promotion in the clinical teaching of joint surgery.

CD72 gene expression in immune thrombocytopenia.

To explore the role of CD72 in the pathogenesis of immune thrombocytopenia (ITP), we detected CD72, Sema4D, IL-2, IL-4, and IFN-γ mRNA expressions and the levels of plasma Sema4D, IL-2, IL-4, IL-6, and IFN-γ in ITP patients (n = 39) and controls (n = 23). The levels of plasma IL-2, IL-4, and IL-6 were assayed by radioimmunoassay, and the levels of plasma IFN-γ and Sema4D were analyzed by enzyme-linked immunosorbent assay. Sema4D, CD72, IL-2, IFN-γ, and IL-4mRNA expressions were analyzed by real-time quantitative reverse-transcription polymerase chain reaction. The expression of CD72 mRNA in ITP patients (n = 23) with active disease was significantly lower than that in patients in remission (p = 0.029) (n = 16) and controls (p = 0.0296) (n = 23). The IFN-γ/IL-4 mRNA (Th1/Th2) expression in ITP patients with active disease and in remission was significantly higher than that in controls (p = 0.0023, p = 0.0125, respectively). The expression of IL-2 mRNA in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0418) and controls (p = 0.004). The level of plasma IL-2 in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0029) and controls (p = 0.0101). The levels of plasma IL-4 in ITP patients with active disease and in remission were significantly higher than that of controls (p = 0.0093, p = 0.0053, respectively). CD72 mRNA expression level might correlate with Sema4D mRNA expression in peripheral blood mononuclear cells and level of plasma IL-2 in active ITP patients (p = 0.024 and p = 0.036). Our findings suggest that CD72 might be involved in the pathophysiological process of the ITP disease by increasing B-cell receptor signals.

Effects of CD70 and CD11a in immune thrombocytopenia patients.

INTRODUCTION: CD70 and CD11a are co-stimulatory molecules that are important for the immune functions of T, B lymphocytes. Over-expressions of CD70 or CD11a cause T cell to be autoreactive. OBJECTIVES: The purpose of this study was to explore the effect of CD70 and CD11a in immune thrombocytopenia (ITP). METHODS: CD70 and CD11a mRNAs and protein expressions in CD4(+) T cells from ITP patients were measured respectively by real-time quantitative-PCR (RT-PCR) and flow cytometry. The apoptosis of T cells, B cells, and platelets in the PBMCs were analyzed by flow cytometry, and secretion of IL-4, IFN-γ, as well as IgG in the reaction supernatant were detected by ELISA. In order to investigate the effects of CD70 and CD11a over-expression on pathogenesis of ITP, anti-CD70, and anti-CD11a mAbs were used to block the signaling pathways. RESULTS: CD70 and CD11a mRNAs and protein expressions in CD4(+) T cells from ITP patients were significantly higher than healthy controls. In vitro co-culturing of PBMCs with anti-CD70 or anti-CD11a, the apoptosis of T, B lymphocytes were significantly increased but apoptosis of platelets were reduced. Anti-CD11a and anti-CD70 both significantly suppressed the secretion of IFN-γ, while anti-CD11a significantly promoted the secretion of IL-4. There was no significant difference in the healthy group. CONCLUSIONS: CD70 and CD11a facilitate the survival of T, B lymphocytes and indirectly enhance the destruction of platelets in ITP. Blockade of CD70 or CD11a are promising therapeutic approaches for ITP.

Association of cytotoxic T-lymphocyte antigen 4 gene polymorphisms with idiopathic thrombocytopenic purpura in a Chinese population.

Idiopathic thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune hemorrhagic disease with many immune dysfunctions. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T-lymphocyte surface molecule that can down modulate and terminate immune responses. Recently, several studies have confirmed that some polymorphisms of this gene can influence its expression level, therefore speculating that they might be associated with autoimmune diseases. In order to investigate the role of the CTLA-4 gene in ITP, we investigated -318 and CT60 polymorphisms of the CTLA-4 gene in 186 ITP patients and 162 healthy controls through polymerase chain reaction (PCR)-restriction fragment length polymorphism. No significant differences were revealed in genotypes and allele distributions between the patients with ITP and the controls in both sites. Similar results were observed between the two groups when stratified by first onset age and disease course including acute childhood, chronic childhood, acute adult, and chronic adult. In the conclusion, these two single-nucleotide polymorphisms in CTLA-4 are not associated with susceptibility to ITP in a Chinese population.

Anticonvulsant effect of Rhynchophylline involved in the inhibition of persistent sodium current and NMDA receptor current in the pilocarpine rat model of temporal lobe epilepsy.

Rhynchophylline (RIN) is a significant active component isolated from the Chinese herbal medicine Uncaria rhynchophylla. Several studies have demonstrated that RIN has a significant anticonvulsant effect in many types of epilepsy models in vivo. However, the mechanisms of the anticonvulsant effect remain elusive. Using combined methods of behavioral testing, immunofluorescence and electrophysiological recordings, we characterized the anticonvulsant effect of RIN in a pilocarpine-induced status epilepticus (SE) rat model of temporal lobe epilepsy (TLE) and investigated the underlying cellular mechanisms. In one set of experiments, rats received RIN treatment prior to pilocarpine injection. In a second set of experiments, rats received RIN treatment following the onset of stage 3 seizures. Pretreatment and posttreatment with RIN effectively reduced the seizure severity in the acute phase of TLE. Furthermore, RIN protected medial entorhinal cortex (mEC) layer III neurons from neuronal death and terminated spontaneous epileptiform discharge of mEC layer II neurons in SE-experienced rats. Whole-cell voltage-clamp recordings indicated that RIN inhibited neuronal hyperexcitability via inhibition of the persistent sodium current (INaP) and NMDA receptor current. Immunofluorescence experiments also demonstrated that RIN rectified the pilocarpine-induced upregulation of Nav1.6 and NR2B protein expression. In conclusion, our results identified RIN as an anticonvulsant agent that inhibited ictal discharge via INap and NMDA receptor current inhibition.

Arousal effects of orexin A on acute alcohol intoxication-induced coma in rats.

The key role of the hypothalamic neuropeptides orexins in maintenance and promotion of arousal has been well established in normal mammalian animals, but whether orexins exert arousal effects under pathological condition such as coma was little studied. In this study, a model of unconscious rats induced by acute alcohol intoxication was used to examine the effects of orexins through intracerebroventricular injection. The results revealed that either orexin A or orexin B induced decrease of duration of loss of right reflex in alcohol-induced unconscious rats. In the presence of the selective orexin receptor 1 antagonist SB 334867 and orexin receptor 2 antagonist TCS OX2 29, the excitatory action of orexin A was completely blocked. Our data further presented that orexin A also induced reduction of delta power in EEG in these rats. Single-unit recording experiment in vivo demonstrated that orexin A could evoke increase of firing activity of prefrontal cortex neurons in unconscious rats. This excitation was completely inhibited by an H(1) receptor antagonist, pyrilamine, whereas application of α(1)-adrenoreceptor antagonist prazosin or 5-HT(2) selective receptor antagonist ritanserin partially attenuated the excitatory effects of orexin A on these neurons. Consistently, the results of EEG recordings showed that microinjection of pyrilamine, prazosin, or ritanserin suppressed reduction of delta power in EEG induced by orexin A on unconscious rats. Thus, these data suggest that orexins exert arousal effects on alcohol-induced unconscious rats by the promotion of cortical activity through activation of histaminergic, noradrenergic and serotonergic systems. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Increased expressions of DNA methyltransferases contribute to CD70 promoter hypomethylation and over expression of CD70 in ITP.

CD70 (TNFSF7), as a methylation susceptive immune gene, was hypomethylated in some autoimmune diseases. To investigate the status of CD70 methylation and the expressions of genes that regulated methylation in immune thrombocytopenia (ITP) patients, the expressions of CD70 mRNA and protein in CD4(+) T cells from ITP and controls were measured respectively by real-time PCR and flow cytometry. Methylation specific high resolution melting (MS-HRM) technology was applied to detect CD70 promoter methylation indices. Transcription levels of DNA methyltransferases (DNMTs), methylated CpG binding protein 2 (MBD2) were measured. The results showed that CD70, DNMTs and MBD2 was over expressed and methylation indices of CD70 promoter in CD4(+) T cells from ITP patients were lower than that from healthy controls. The transcription levels of CD70 showed significantly inverse correlation with methylation indices in ITP patients but significantly positive correlation with that of DNMTs. We concluded that DNMTs functioned as demethylases as MBD2, while increased DNMTs and MBD2 may cause demethylation and over expression of CD70 in CD4(+) T cells, potentially contributing to the pathogenesis of ITP.

Short-term sleep deprivation increases intrinsic excitability of prefrontal cortical neurons.

Short-term sleep deprivation (SD) has been shown to enhance cortical activity. However, alterations in the cellular excitability of cortical neurons following SD are not yet fully understood. The present study investigated the effects of 4-hour SD on pyramidal neurons in the prefrontal cortex (PFC) of rats using whole-cell patch-clamp recording. SD led to an increase in the initial slope of firing frequency-current curve and a decrease in frequency adaptation, which were reversed by recovery sleep (RS). Correspondingly, the total afterhyperpolarization (AHP) was reduced in the SD group and returned in the RS group. Furthermore, the component of AHP changed after SD seemed to be sensitive to Ca(2+). These observations indicate an enhancement in intrinsic excitability due to short-term SD, and suggest a role for Ca(2+)-dependent AHP in this change. The findings of the present study may provide a possible explanation for the SD-induced increase in cortical activity.