Improving the tumor selectivity of T cell engagers by logic-gated dual tumor-targeting.

Targeting single tumor antigens makes it difficult to provide sufficient tumor selectivity for T cell engagers (TCEs), leading to undesirable toxicity and even treatment failure, which is particularly serious in solid tumors. Here, we designed novel trispecific TCEs (TriTCEs) to improve the tumor selectivity of TCEs by logic-gated dual tumor-targeting. TriTCE can effectively redirect and activate T cells to kill tumor cells (∼18 pM EC50) by inducing the aggregation of dual tumor antigens, which was ∼70- or 750- fold more effective than the single tumor-targeted isotype controls, respectively. Further in vivo experiments indicated that TriTCE has the ability to accumulate in tumor tissue and can induce circulating T cells to infiltrate into tumor sites. Hence, TriTCE showed a stronger tumor growth inhibition ability and significantly prolonged the survival time of the mice. Finally, we revealed that this concept of logic-gated dual tumor-targeted TriTCE can be applied to target different tumor antigens. Cumulatively, we reported novel dual tumor-targeted TriTCEs that can mediate a robust T cell response by simultaneous recognition of dual tumor antigens at the same cell surface. TriTCEs allow better selective T cell activity on tumor cells, resulting in safer TCE treatment.

[ORMDL3 polymorphisms and their relationship with OPN and TGF-ß1 levels in children with asthma in Hunan, China: an analysis of 98 cases].

OBJECTIVE: To investigate ORMDL3 polymorphisms in children with asthma in Hunan, China, and to determine the relationship between ORMDL3 polymorphisms and serum osteopontin (OPN) and transforming growth factor-ß1 (TGF-ß1) levels. METHODS: Peripheral blood samples were collected in children with asthma (n=98; astma group) or without asthma (n=30; control group) from Hunan, China. The asthma group was subdivided into atopic (n=62) and non-atopic (n=36) subgroups. Single nucleotide polymorphism (SNP) analysis was performed, and serum OPN and TGF-ß1 levels were measured. RESULTS: There were no significant differences in genotype and allele frequencies of rs7216389 of the ORMDL3 gene between the asthma and control groups. The serum level of OPN in the asthma group was significantly higher than in the control group (P<0.05). Both the atopic and non-atopic subgroups showed increased serum levels of OPN compared with the control group (P<0.05). The serum level of TGF-ß1 in the atopic subgroup was significantly higher than in the control group (P<0.05). The serum levels of OPN and TGF-ß1 showed no significant differences in asthmatic children with different genotypes. The serum levels of OPN and TGF-ß1 were in a positive linear correlation in the asthma group (r=0.620; P<0.01) and its two subgroups (r=0.734, 0.649 respectively; P<0.01). CONCLUSIONS: In children from Hunan, China, the SNP (rs7216389) of ORMDL3 is not related to asthma susceptibility. OPN and TGF-ß1 may be involved in the development of asthma, and they are in a positive linear correlation. The SNP (rs7216389) of ORMDL3 does not influence the expression of OPN and TGF-ß1, suggesting that it may not be associated with airway remodeling.

Diagnostic values of soluble triggering receptor expressed on myeloid cells (sTREM-1) and interferon-inducible protein-10 (IP-10) for severe mycoplasma pneumoniae pneumonia in children.

OBJECTIVE: Early diagnosis of Severity Mycoplasma Pneumoniae Pneumonia (SMPP) has been a worldwide concern in clinical practice. Two cytokines, soluble Triggering Receptor Expressed on Myeloid cells (sTREM-1) and Interferon-Inducible Protein-10 (IP-10), were proved to be implicated in bacterial infection diseases. However, the diagnostic value of sTREM-1 and IP-10 in MPP was poorly known. This study aimed to investigate the diagnostic value of sTREM-1 and IP-10 for SMPP. METHODS: In this prospective study, the authors enrolled 44 children with MPP, along with their clinical information. Blood samples were collected, and cytokine levels of sTREM-1 and IP-10 were detected with ELISA assay. RESULTS: Serum levels of sTREM-1 and IP-10 were positively correlated with the severity of MPP. In addition, sTREM-1 and IP-10 have significant potential in the diagnosis of SMPP with an Area Under Curve (AUC) of 0.8564 (p-value = 0.0001, 95% CI 0.7461 to 0.9668) and 0.8086 (p-value = 0.0002, 95% CI 0.6918 to 0.9254) respectively. Notably, the combined diagnostic value of sTREM-1 and IP-10 is up to 0.911 in children with SMPP (p-value < 0.001, 95% CI 0.830 to 0.993). CONCLUSIONS: Serum cytokine levels of sTREM-1 and IP-10 have a great potential diagnostic value in children with SMPP.

[Mycoplasma pneumoniae infection and drug resistance in children: an analysis of 1026 cases].

OBJECTIVE: To investigate the Mycoplasma pneumoniae (MP) infection and drug resistance in children with respiratory tract infection and to provide a rational basis for the clinical diagnosis and treatment of MP infection. METHODS: Throat swabs were collected from 3529 children with respiratory tract infection, who visited the pediatric outpatient department or received treatment in the pediatric ward of our hospital from September 2010 to September 2011. The swabs were cultured to detect MP. The drug sensitivity of MP to azithromycin, roxithromycin, erythromycin, acetylspiramycin and clarithromycin was evaluated. RESULTS: Of the 3529 children with respiratory tract infection, 1026 (29.07%) were MP-positive. There were cases of MP infection in all four seasons of the year but infection rates in summer and autumn were significantly higher than in spring and winter (P < 0.05). The infection rate in females was higher than in males (30.43% vs 28.32%; P > 0.05). The infection rate was negatively correlated with age in these children, and there were significant differences in the infection rate among all age groups (P < 0.05). For macrolide antibiotics suitable for children, the cultured MP developed the highest resistance to roxithromycin, followed by erythromycin, acetylspiramycin, clarithromycin, and azithromycin, with significant differences among them (P < 0.01). CONCLUSIONS: MP infection rate is very high among children with respiratory tract infection. The incidence of MP infection is relatively low among school-age children and children are more susceptible to MP infection in summer and autumn than in spring and winter. Throat swabs should be cultured and drug sensitivity tests should be performed as early as possible in children with respiratory tract infection, so that proper intervention can be undertaken in time to reduce drug-resistant strains of MP.

[Features of pathological changes in the non-myelin sheath of rats with experimental autoimmune encephalomyelitis].

OBJECTIVE: To study the pathological changes in the non-myelin sheath by observing histological damages to the neurofilament protein and apoptosis of neurons in rats with experimental autoimmune encephalomyelitis (EAE). METHODS: Forty-eight Wistar rats were randomly divided into two groups: control and EAE (24 rats in each group). Behavioral changes were observed. Inflammation reactions and demyelination were observed by hematoxylin eosin staining and LOYEZ staining.The level of neurofilament was detected by immunohistochemistry. Apoptosis of the neuron in the spinal cord was detected by TUNEL. RESULTS: Behavioral and histological results confirmed that the model of EAE rats was prepared successfully. In the EAE group, typical morphological features of axonal damage (sparsed axonal density, axonal distortion, axonal transection and even axonal disappearance) were found from the seventh day after immunization and the morphological changes were the most obvious on the fourteenth day. Neurofilament density in the EAE group was significantly lower than in the control group (P<0.01) at 7, 14 and 21 days after immunization. The neuronal apoptosis index in the EAE group at 7, 14 and 21 days after immunization was significantly higher than in the control group (P<0.01). CONCLUSIONS: In addition to inflammatory demyelination, axonal damage and neuronal apoptosis can be observed in the early stage of EAE. Pathological changes may be associated with neurological dysfunction.

[Effects of baicalin on apoptosis in rats with autoimmune encephalomyelitis].

OBJECTIVE: To study the therapeutic efficacy of baicalin and its effect on apoptosis of inflammatory cells in spinal cords in Wistar rats with autoimmune encephalomyelitis (EAE). METHODS: Forty-four rats were randomly divided into four groups: normal control group (control, n=10), EAE group (n=12), and two intervention groups with dexamethasone (DXM) or baicalin. Seven days after immunization, the two intervention groups were injected intraperitoneally with DXM (1 mg/kg) and baicalin (200 mg/kg) for 1 week, respectively. The spinal cords were removed 14 days after immunization, and stained with hematoxylin and eosin. MBP expression in spinal cords was detected by immunohistochemistry. The apoptosis of inflammatory cells in spinal cords was detected by TUNEL. RESULTS: The weight gain rate in the untreated EAE and the DXM or baicalin intervention groups were significantly lower than that in the control group (P<0.05). The weight gain rate in the baicalin intervention group was significantly higher than that in the untreated EAE and the DXM intervention groups (P<0.05). The scores of neurological function in the two intervention groups were significantly higher than that in the untreated EAE group (P<0.05). DXM or baicalin treatment significantly increased the MBP expression compared with the untreated EAE group (P<0.05). The apoptosis of inflammatory cells increased more in the DXM and the baicalin intervention groups compared with the untreated EAE groups (P<0.05). CONCLUSIONS: Baicalin has protective effects against EAE in rats. It can promote the apoptosis of inflammatory cells in spinal cords.

Soluble Expression of Fc-Fused T Cell Receptors Allows Yielding Novel Bispecific T Cell Engagers.

The specific recognition of T cell receptors (TCR) and peptides presented by human leukocyte antigens (pHLAs) is the core step for T cell triggering to execute anti-tumor activity. However, TCR assembly and soluble expression are challenging, which precludes the broad use of TCR in tumor therapy. Herein, we used heterodimeric Fc to assist in the correct assembly of TCRs to achieve the stable and soluble expression of several TCRs in mammalian cells, and the soluble TCRs enable us to yield novel bispecific T cell engagers (TCR/aCD3) through pairing them with an anti-CD3 antibody. The NY-ESO-1/LAGE-1 targeted TCR/aCD3 (NY-TCR/aCD3) that we generated can redirect naïve T cells to specific lysis antigen-positive tumor cells, but the potency of the NY-TCR/aCD3 was disappointing. Furthermore, we found that the activation of T cells by NY-TCR/aCD3 was mild and unabiding, and the activity of NY-TCR/aCD3 could be significantly improved when we replaced naïve T cells with pre-activated T cells. Therefore, we employed the robust T cell activation ability of staphylococcal enterotoxin C2 (SEC2) to optimize the activity of NY-TCR/aCD3. Moreover, we found that the secretions of SEC2-activated T cells can promote HLA-I expression and thus increase target levels, which may further contribute to improving the activity of NY-TCR/aCD3. Our study described novel strategies for soluble TCR expression, and the optimization of the generation and potency of TCR/aCD3 provided a representative for us to fully exploit TCRs for the precision targeting of cancers.

Inhibition of insulin resistance by PGE1 via autophagy-dependent FGF21 pathway in diabetic nephropathy.

Insulin resistance is a critical process in the initiation and progression of diabetic nephropathy (DN). Alprostadil (Prostaglandin E1, PGE1) had protective effects on renal function. However, it is unknown whether PGE1 inhibited insulin resistance in renal tubule epithelial cells via autophagy, which plays a protective role in DN against insulin resistance. Insulin resistance was induced by palmitic acid (PA) in human HK-2 cells, shown as the decrease of insulin-stimulated AKT phosphorylation, glucose transporter-4 (GLUT4), glucose uptake and enhanced phosphorylation of insulin receptor substrate 1(IRS-1) at site serine 307 (pIRS-1ser307) and downregulated expression of IRS-1. Along with less abundance of p62, autophagy markers LC3B and Beclin-1 significantly increased in HK-2 cells exposed to PA. Such abnormal changes were significantly reversed by PGE1, which mimicked the role of autophagy gene 7 small interfering RNA (ATG7 siRNA). Furthermore, PGE1 promoted the protein expression of autophagy-related fibroblast growth factor-21 (FGF21), which alleviated insulin resistance. Results from western blotting and immunohistochemistry indicated that PGE1 remarkably restored autophagy, insulin resistance and the FGF21 expression in rat kidney of type 2 diabetes mellitus (T2DM). Collectively, we demonstrated the potential protection of PGE1 on insulin resistance in renal tubules via autophagy-dependent FGF21 pathway in preventing the progression of DN.

Acid Sphingomyelinase Down-regulation Alleviates Vascular Endothelial Insulin Resistance in Diabetic Rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in patients with type 2 diabetes. Acid sphingomyelinase (ASM) is a soluble glycoprotein which plays a vital role in the development and progression of various diseases such as cardiovascular and metabolic diseases. However, it remains unknown if ASM regulates insulin resistance in vascular endothelial cells in type 2 diabetes. ASM down-regulation with gene silencing and selective inhibitor amitriptyline was used in the rat aortic endothelial cells (RAECs) treated with palmitic acid (PA), a common saturated free fatty acid, which is thought to be the major cause of insulin resistance. It was shown that ASM down-regulation increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of pIRS-1-ser307 and AKT-ser473 via ceramide, consequently resulting in the decrease of the production of endothelial nitric oxide synthase (eNOS) and nitric oxide in PA-induced RAECs. We further found that ASM down-regulation blocked the Nox2- and Nox4-dependent superoxide (O2 -· ) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vivo, amitriptyline relieved the vasodilatory response to acetylcholine and restored the level of ceramide, Nox2 and Nox4 in the aorta endothelium of high-fat diet-fed rats following an injection of streptozotocin. Taken together, these results suggest that ASM down-regulation can improve endothelial insulin resistance which is attributed to inhibiting redox signalling in RAECs. Thus, these data support the idea that ASM is a promising clinical biomarker and potential therapeutic target for diabetic vascular complication.

Argirein alleviates vascular endothelial insulin resistance through suppressing the activation of Nox4-dependent O2- production in diabetic rats.

BACKGROUND: Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in type 2 diabetes mellitus (T2DM). Therefore, there are great potential clinical implications in developing pharmacological interventions targeting endothelial insulin resistance. Our previous studies indicated that argirein which was developed by combining rhein with L-arginine by a hydrogen bond, could substantially relieved stress related exacerbation of cardiac failure and alleviated cardiac dysfunction in T2DM, which was associated with suppressing NADPH oxidase activity. However, it is unclear whether argirein treatment attenuates the vascular lesion and dysfunction in T2DM and its underlying mechanisms. METHODS AND RESULTS: The rat aortic endothelial cells (RAECs) were used to treat with palmitic acid (PA), a most common saturated free fatty acid, which could induce insulin resistance. It was showed that argirein increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of IRS-1-ser307 and AKT-ser473, consequently resulting in the increase of the production of eNOS and NO in PA-induced RAECs. We further found that argirein blocked the Nox4-dependent superoxide (O2-.) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vitro, argirein increased the release of endothelial NO to relieve the vasodilatory response to acetylcholine and insulin, and restored the expression of Nox4 and pIRS-1-ser307 in the aorta endothelium of high-fat diet (HFD)-fed rats following an injection of streptozocin (STZ). CONCLUSION: These results suggested that argirein could improve endothelial insulin resistance which was attributed to inhibiting Nox4-dependent redox signaling in RAECs. These studies thus revealed the novel effect of argirein to prevent the vascular complication in T2DM.

[Protective effects of heat shock preconditioning on the experimental autoimmune encephalomyelitis rats].

OBJECTIVE: To study the effects of heat shock preconditioning on the expression of heat shock protein-70 (HSP70) and apoptosis of the neuron in experimental autoimmune encephalomyelitis (EAE) rats. METHODS: Thirty-six Wistar rats were randomly divided into control, EAE and heat shock preconditioning groups (n=12 each). The EAE animal model was induced with guinea pig myelin basic protein. Heat shock preconditioning was performed 24 hrs prior to the EAE model inducement. No treatment was done in the control group. The neurological signs were observed after immunization. The spinal cords were removed and stained with hematoxylin and eosin. HSP70 was detected by immunohistochemistry. Apoptosis of the neuron was measured by TUNEL. RESULTS: Heat shock preconditioning significantly alleviated clinical signs and neuronal injury. HSP70 expression in the heat shock preconditioning group was significantly higher than in the untreated EAE group (21.08 +/- 0.87 vs 10.17 +/- 0.51; P < 0.01). Heat shock preconditioning suppressed apoptosis of the neuron compared with the EAE group (apoptosis rate: 21.92 +/- 1.00% vs 58.92 +/- 1.67%; P < 0.01). CONCLUSIONS: Heat shock preconditioning might improve the neurological outcome in EAE rats, possibly through the induction of HSP70 synthesis and the reduction of apoptosis of the neuron in spinal cords.

Protection by simvastatin on hyperglycemia-induced endothelial dysfunction through inhibiting NLRP3 inflammasomes.

Recent studies have demonstrated that NLRP3 inflammasome complex acts as pivotal elements to initiate inflammatory responses and plays an important role in the dysfunction of cardiovascular complications. Meanwhile, simvastatin prevents vascular endothelial dysfunction from inflammasome invasion contributing to reduce cardiovascular risk. However, Whether or not the simvastatin improves vascular endothelial barrier function through inhibiting the activation of NLRP3 inflammasome pathway remains unknown. Here, we explored the role and mechanisms of simvastatin in the activation of NLRP3 inflammasome which are involved in vascular endothelial hyperpermeability causing by the disruption of tight junction protein ZO-1 and adherens junction protein VE-Cadherin, an early initiation of cardiovascular complication. Our results found that high glucose significantly induced the formation and activation of NLRP3 inflammasome through NADPH oxidase-dependent reactive oxygen species (ROS) formation, associated with vascular endothelial hyperpermeability causing by ZO-1 and VE-Cadherin disruption in the rat aortic endothelial cells (RAECs). Simvastatin treatment remarkably abolished vascular endothelial hyperpermeability and enhanced the protein expression of ZO-1 and VE-Cadherin through NLRP3 inflammasome. Mechanistically, the inhibitory role of simvastatin endothelial hyperpermeability is attributed to the decreased release of cytoplasmic high mobility group box protein-1 (HMGB1) derived from endothelial NLRP3 inflammasome activation. We further confirm the protective role of simvastatin on vascular leakage in the heart of diabetic rats injected with Evans blue dye, which was associated with HMGB1 release in the serum. Collectively, the mechanism of simvastatin treatment alleviating vascular endothelial permeability dysfunction may be through inhibiting the NLRP3 inflammasome-dependent HMGB1 release in RAECs.