Analysis of T-lymphocyte subsets and risk factors in children with tuberculosis.

BACKGROUND: Tuberculosis (TB) is not only related to infection but also involves immune factors. This study explores the changes in T-lymphocyte subsets in children with TB who are human immunodeficiency virus (HIV)-negative and examines their relationship using chest computed tomography (CT) scans. Additionally, the study identifies risk factors for severe TB (STB) in children and establishes relevant risk prediction models. METHODS: We recruited 235 participants between 2018 and 2022, comprising 176 paediatric patients with TB who were HIV-negative and 59 age-matched children with bacterial community-acquired pneumonia (CAP). We quantitatively analysed and compared T-lymphocyte subsets between the two groups and among different types of TB infection. Both univariate and multivariate analyses of clinical and laboratory characteristics were conducted to identify independent risk factors for STB in children and to establish a risk prediction model. RESULTS: The absolute counts of CD3, CD4 and CD8 T-cells in children with TB infection decreased significantly compared with bacterial CAP. The percentage of CD8 T-cells increased, whereas the percentage of CD4 T-cells did not change significantly. The absolute count of CD3, CD4 and CD8 T-cells in extrapulmonary TB (EPTB) was significantly higher than in extra-respiratory TB, with unchanged subset percentages. According to chest CT lesion classification, CD4 T-cell counts decreased significantly in S3 compared with S1 or S2, with no significant change in CD3 and CD8 T-cell counts and percentages. No significant differences were observed in lymphocyte subset counts and percentages between S1 and S2. Univariate analyses indicated that factors such as age, symptom duration, white blood cell count, platelet count, neutrophil-to-lymphocyte ratio (NLR), erythrocyte sedimentation rate, prealbumin level, albumin level, globulin level, albumin/globulin (A/G) ratio, high-sensitivity C-reactive protein (Hs-CRP) level and CD4 and CD8 T-cell counts are associated with STB. Multivariate logistic regression analysis revealed that age, Hs-CRP level, NLR, symptom duration and A/G ratio are independent risk factors for STB in children. Increased age, Hs-CRP levels and NLR, along with decreased A/G, correlate with increased susceptibility to STB. A nomogram model, based on these independent risk factors, demonstrated an area under the receiver operating characteristics curve of 0.867 (95% CI: 0.813-0.921). Internal verification confirmed the model's accuracy, with the calibration curve approaching the ideal and the Hosmer-Lemeshow goodness-of-fit test showing consistent results (χ2 = 12.212, p = 0.142). CONCLUSION: In paediatric patients with TB, the absolute counts of all lymphocyte subsets were considerably reduced compared with those in patients with bacterial CAP. Clinicians should consider the possibility of EPTB infection in addition to respiratory infections in children with TB who have higher CD3, CD4 and CD8 T-cell counts than the ERTB group. Furthermore, CD4 T-cell counts correlated closely with the severity of chest CT lesions. Age, symptom duration, A/G ratio, Hs-CRP level and NLR were established as independent risk factors for STB. The nomogram model, based on these factors, offers effective discrimination and calibration in predicting STB in children.

Interleukin-35 Modulates the Imbalance Between Regulatory T Cells and T Helper 17 Cells in Enterovirus 71-Induced Hand, Foot, and Mouth Disease.

Interleukin (IL)-35 modulates the imbalance between regulatory T cells (Tregs) and T helper (Th) 17 cells, which played vital roles in the pathogenesis of autoimmune and infectious diseases. However, the role of Tregs/Th17 cell imbalance and the regulatory functions of IL-35 have remained largely unknown in enterovirus 71 (EV71)-induced hand, foot, and mouth disease (HFMD). In this study, a total of 47 HFMD patients (30 with mild HFMD and 17 with severe HFMD) and 13 healthy individuals were enrolled. The frequencies of CD4+CD25+CD127dim/- Tregs and CD4+IL-17+ Th17 cells, as well as IL-35 expression levels, were measured. Cellular proliferation and cytokine production was also determined in purified Tregs following recombinant IL-35 stimulation. An imbalance between Tregs and Th17 cells was observed in children with severe HFMD, which manifested as a reduction in the Tregs population and an elevation in the Th17 population. Serum IL-35 concentrations were also decreased in case of severe HFMD, which correlated with the Tregs:Th17 cell ratios. Recombinant IL-35 stimulation increased the proportion of Tregs, but downregulated that of Th17 cells. Treatment with IL-35 enhanced Tregs suppressive function and IL-35 and IL-10 expression, but reduced IL-22 secretion in both healthy individuals and those with severe HFMD. The Tregs:Th17 cell ratio was increased in the convalescent patients, however, a significant reduction in serum IL-35 was not observed. Our findings indicated that EV71 infection shifted the Tregs:Th17 cell ratio through IL-35 by downregulating inhibitory cytokine production and reducing the cell-to-cell contact inhibition of effector T cells. Regulation of IL-35 as it relates to the Tregs/Th17 balance may play a critical role in the pathogenesis of EV71-associated HFMD.

Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis / Isoflurano fornece neuroproteção em lesão cerebral hipóxico-isquêmica neonatal por inibição da apoptose

Abstract Background and objectives: Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. Methods: A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. Results: Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. Conclusions: Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis.

Resumo Justificativa e objetivos: Isoflurano é um éter volátil halogenado usado para anestesia por via inalatória. É amplamente usado na clínica como um anestésico para inalação. A lesão hipóxico-isquêmica neonatal ocorre no cérebro imaturo e resulta em morte celular tardia via excitotoxicidade e estresse oxidativo. Isoflurano mostrou ter propriedades neuroprotetoras que formam uma base benéfica para o seu uso tanto em cultura de células quanto em modelos animais, incluindo vários modelos de lesão cerebral. Nosso objetivo foi determinar o efeito neuroprotetor de isoflurano em hipóxia cerebral e elucidar o mecanismo subjacente. Métodos: Fatias de hipocampo, em fluido cerebrospinal artificial (CSFA) com glicose e privação de oxigênio, foram usadas como um modelo in vitro de hipóxia cerebral. O pico de população ortodrômica (PPO) e o potencial de lesão hipóxica (PLH) foram registrados nas regiões CA1 e CA3. A concentração de neurotransmissores de aminoácidos na solução de perfusão das fatias de hipocampo foi medida. Resultados: O tratamento com isoflurano retardou a eliminação do PPO e melhorou a recuperação do PPO; diminuiu a frequência do PLH, retardou o início do PLH e aumentou a duração do PLH. O tratamento com isoflurano também diminuiu a liberação de neurotransmissores de aminoácidos induzida pela hipóxia, como aspartato, glutamato e glicina, mas os níveis de ácido γ-aminobutírico (GABA) estavam elevados. Estudos morfológicos mostram que o tratamento de edema com isoflurano atenuou o edema de neurônios piramidais na região CA1. Também reduziu a apoptose, como mostrado pela expressão reduzida da caspase-3 e genes PARP. Conclusões: Isoflurano mostrou um efeito neuroprotetor na lesão neuronal no hipocampo induzida por hipóxia através da supressão de apoptose.

Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis.

BACKGROUND AND OBJECTIVES: Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. METHODS: A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. RESULTS: Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. CONCLUSIONS: Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis.

[Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis]. / Isoflurano fornece neuroproteção em lesão cerebral hipóxico­isquêmica neonatal por inibição da apoptose.

BACKGROUND AND OBJECTIVES: Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. METHODS: A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. RESULTS: Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. CONCLUSIONS: Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis.

[Free radial forearm flap for reconstruction of head and neck soft tissue defects after tumor resection].

OBJECTIVE: To explore the clinical effect of the free radial forearm flap on repairing tissue defects and reconstructing functions after tumor resection.
 METHODS: From January, 2003 to December, 2011, 70 patients, including 43 squamous cell carcinomas of tongue, 12 buccal cancers, 5 carcinomas of the soft palate, 4 basal cell carcinomas of external nose, 3 lower lip cancers, 2 upper lip cancers, and 1 posterior wall of hypopharynx carcinoma, with the soft tissue defects in the head and neck underwent reconstructive operations with the free radial forearm flap after the malignant tumor resection. The area of defects ranged from 5 cm × 4 cm to 14 cm × 8 cm with the process of diseases from 4 to 30 months. The technique for grafting the free radial forearm flap and the appearance at sites of the donor and recipient, and the influence on the anatomy and function in both local sites were analyzed.
 RESULTS: In the 70 patients, only 1 case of flap appeared necrosis due to venous reflux obstacle, and the remaining (98.4 ﹪) survived. During the follow-up for 12-36 months, one case of hypopharyngeal carcinoma died from distant metastasis a year later, 2 cases of tongue cancer died of cardiovascular accident. Morphology and function for the sites at donor and recipient were satisfactory.
 CONCLUSION: Free radical forearm flap is a good choice for the repair and functional reconstruction for tissue defects after tumor resection.

Expression of CBY and methylation of CBY at promoter region in human laryngeal squamous cell carcinoma.

AIMS AND BACKGROUND: Chibby (CBY), a ß-catenin binding partner, inhibits Wnt/ß-catenin-mediated transcriptional activation by competing with Tcf/Lef factors for ß-catenin binding and promoting the export of ß-catenin from nucleus to cytoplasm. The regulatory effect of CBY in this signaling pathway suggests its biological importance as a potential tumor suppressor gene. The purposes of this study were to determine whether the expression of CBY was downregulated in human laryngeal squamous cell carcinoma (LSCC) samples, the CpG sites of CBY at the promoter region were methylated in these tumor samples, and reduced expression of CBY was induced by methylation of CBY promoters. METHODS: CBY expression was investigated by quantitative real-time PCR and immunohistochemistry in samples from 36 LSCC patients. The methylation status of the CBY promoter was detected by methylation-specific PCR. RESULTS: Compared with normal laryngeal mucosa, the expression of CBY was downregulated in LSCC samples. The reduced CBY expression rate was 58.33% (21/36) at the mRNA and 66.67% (24/36) at the protein level. The promoters of CBY were methylated in 12/36 tumor samples, partially methylated in 5, and unmethylated in 19 samples. The methylation rate including incomplete methylation was 47.22% (17/36) in tumor samples, while no methylation was detected in normal laryngeal squamous epithelium. Compared with the unmethylated group, the expression of CBY was significantly different in the methylated group (p<0.05) but similar in the partially methylated group (p>0.05). CONCLUSIONS: Our data indicate that CBY expression was downregulated in LSCC, which may be partially caused by methylation of CBY promoters.

Downregulated Chibby in laryngeal squamous cell carcinoma with increased expression in laryngeal carcinoma Hep-2 cells.

Chibby (Cby) inhibits Wnt/ß-catenin-mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of ß-catenin) to bind to ß-catenin. This suggests that Cby could be a tumor suppressor protein. In the present study, we examined Cby expression in laryngeal squamous cell carcinoma (LSCC) and its function and mechanism in laryngeal carcinoma cell lines. Cby expression levels were investigated by immunohistochemistry in a panel of 36 LSCC patient cases. The expression of ß-catenin, c-myc and cyclin D1 in Hep-2 were determined through RT-PCR and western blot analysis. Activity of Wnt/ß-catenin signaling pathway after overexpression of Cby was measured by TCF/LEF luciferase reporter gene assay. Proliferation, clone forming ability, cell cycle distribution and cell apoptosis of Hep-2 cells were detected by MTT assay, plate colony forming assay, flow cytometry and TUNEL assay, respectively. This study showed that expression of Cby protein was strongly downregulated in LSCC tumor tissues in comparison to normal laryngeal mucosa samples. No significant correlation was found between the expression of Cby in tumor tissue and gender, age, clinical stage and tumor differentiation of laryngeal cancer patients. When Cby was overexpressed in Hep-2 cells, the expression of cyclin D1 was reduced and ß-catenin activity was inhibited. Proliferation and plate colony forming assays revealed a significant inhibitory effect of Cby on growth and colony formation ability of Hep-2 cells after Cby overexpression in comparison to control and mock-infected cells. In addition, we also found that upregulated expression of Cby resulted in accumulation of numbers of cells in G0/G1 phase with concomitant decrease in S phase by cell cycle assay. TUNEL staining demonstrated that, compared with the control group, the rate of apoptosis in the plv-cs2.0-Cby group was significantly increased. Taken together, downregulation of Cby was observed in LSCC, but with no significant correlation to the clinicopathological features of LSCC patients. Overexpression of Cby effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. A better understanding of the mechanisms of Cby gene activation in LSCC could provide potential novel therapeutic targets for human laryngeal carcinoma.

[Change in plasma H2S level and therapeutic effect of H2S supplementation in tubulointerstitial fibrosis among rats with unilateral ureteral obstruction].

OBJECTIVE: To observe the level in plasma hydrogen sulfide (H2S) and the expression of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) (two key synthetases for endogenous H2S generation in the kidney) in obstructed kidney tissue among rats with tubulointerstitial fibrosis (TIF) induced by unilateral ureteral obstruction (UUO), and to explore the role of H2S in TIF. METHODS: Ninety-six male Sprague-Dawley rats were randomly divided into sham-operated, model, low-dose NaHS and high-dose NaHS groups (n=24 each). TIF was induced by UUO in the model, low-dose NaHS and high-dose NaHS groups. The low-dose and high-dose NaHS groups were intraperitoneally injected with NaHS (1.4 and 7.0 µmol/kg respectively) twice daily immediately after operation, and the sham-operated and model groups were intraperitoneally injected with an identical volume of normal saline. In each group, 8 rats were randomly selected and sacrificed at 7, 14 or 21 days after operation. Plasma H2S concentration was measured by deproteinization. The obstructed kidney tissue was subjected to hematoxylin and eosin staining and Masson staining, and the renal tubulointerstitial injury was evaluated under a microscope. mRNA and protein expression of CBS and CSE in the obstructed kidney tissue was measured by RT-PCR and immunohistochemistry respectively. RESULTS: The degree of UUO-induced renal tubulointerstitial injury was negatively correlated with plasma H2S concentration in (r=-0.891, P<0.01). With H2S supplementation, renal tubulointerstitial injury was reduced (P<0.01), the expression of mRNA and protein of CBS and CSE in the kidney tissue and plasma H2S level were upregulated (P<0.01), and the degree of TIF was reduced (P<0.01). There were no significant differences in plasma H2S level and mRNA and protein expression of CBS and CSE between the low-dose and high-dose NaHS groups (P>0.05). CONCLUSIONS: H2S is involved in the development of UUO-induced TIF, and the CBS/H2S and CSE/H2S systems play key roles in this process. H2S supplementation can delay the progression of TIF.

[Correlation between the expression of cytokeratin-18-mRNA and invasion and metastasis of oral squamous cell carcinoma].

OBJECTIVE: To investigate the relationship between the expression of cytokeratin (CK)-18 and biological behavior of oral squamous cell carcinoma (OSCC). METHODS: Twenty-three patients with OSCC were investigated for the expression of CK-18-mRNA by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Correlations between clinical stages, pathological differentiation, lymphatic metastasis and the expression of CK-18-mRNA were evaluated. CK-18-mRNA expression of peripheral blood from the 23 patients and 23 healthy people were also examined. During follow-up after operation, the peripheral blood was collected again for the expression of CK-19-mRNA. RESULTS: Expression of CK-18-mRNA was found in 16 patients. The expression of CK-18-mRNA was significantly associated with clinical stages, tumor differentiation and lymphatic metastasis. CK-18-mRNA was positive in 4 of 23 blood specimens before operation, but during follow-up only 1 of 23 patients was still positive in peripheral blood. CONCLUSIONS: CK-18 may provide additional information in forecasting the metastasis of OSCC and serve as a reference in monitoring recurrence.