IL-1β and IL-17 in cutaneous lupus erythematous skin biopsies: could immunohistochemicals indicate a tendency towards systemic involvement?

Abstract Background: Only a fraction of patients with cutaneous lupus erythematosus (CLE) will eventually progress toward systemic disease (SLE). Objective: To find inflammatory biomarkers which could predict the progression of cutaneous lupus erythematosus (CLE) into systemic lupus erythematosus (SLE) using immunohistochemical (IHC) assays. Methods: Immunohistochemical markers for cytotoxic, inflammatory, and anti-inflammatory responses and morphometric methods were applied to routine paraffin sections of skin biopsies, taken from lesions of 59 patients with discoid lupus, subacute lupus, and lupus tumidus. For the diagnosis of SLE, patients were classified by both the American College of Rheumatology (ACR-82) and the Systemic Lupus International Collaborating Clinics (SLICC-12) systems. Results: Skin samples from CLE/SLE +patients presented higher expression of IL-1β (ARC-82: p = 0.024; SLICC-12: p = 0.0143) and a significantly higher number of cells marked with granzyme B and perforin (ARC: p = 0.0097; SLICC-12: p = 0.0148). Biopsies from CLE/SLE- individuals had higher expression of IL-17 (ARC-82: p = 0.0003; SLICC-12: p = 0.0351) and presented a positive correlation between the density of granzyme A+and FoxP3+ cells (ARC-82: p = 0.0257; SLICC-12: p = 0.0285) and CD8+ cells (ARC-82: p = 0.0075; SLICC-12: p = 0.0102), as well as between granulysin-positive and CD8+ cells (ARC-82: p = 0.0024; SLICC-12: p = 0.0116). Study limitations: Patients were evaluated at a specific point in their evolution and according to the presence or not of systemic disease. The authors cannot predict how many more, from each group, would have evolved towards SLE in the following years. Conclusions: In this cohort, immunohistochemical findings suggested that patients with a tendency to systemic disease will show strong reactivity for IL-1β, while those with purely cutaneous involvement will tend to express IL-17 more intensely.

Molecular detection of IL-10 level to determine severity of periodontitis in type 2 Diabetes Mellitus patients / Detecção molecular do nível de IL-10 para determinar a gravidade da periodontite em pacientes com Diabetes Mellitus tipo 2

Objective: The prevalence of periodontal disease is increasing in most countries including developing and developed countries. It affects 20-50-% of the global population. Patients with type 2 Diabetes Mellitus (DM) with severe periodontal disease had a 3.2 times higher risk of death than individuals without periodontitis. Periodontitis contributes to small-scale systemic inflammation. The objective of this study was to determine the severity of periodontitis using IL-10 (Interleukin-10) level in type 2 diabetes mellitus. Materials and Methods: This study was cross-sectional. All methods were performed following the guidelines and regulations of the Ethics Committee, Faculty of Dental Medicine, Universitas Airlangga. The samples were 90 subjects. The instruments used were questionnaires, periodontal status measurements based on Community Periodontal Index (CPI), and random blood glucose measurements. Data on the IL-10 level was obtained using Gingival Crevicular Fluid (GCF). Results: There was a significant difference in lifestyle in each group. The highest IL-10 level was found in the periodontitis group, followed by the periodontitis with the type 2 DM group. Conclusion: The level of IL-10 can be used to determine periodontitis severity in type 2 DM. Most respondents with the highest level of IL-10 were found in periodontitis followed by periodontitis with type 2 DM group. High levels of IL-10 will decrease the synthesis of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-6 (IL-6), activation of macrophages, and Polymorphonuclear neutrophil (PMN) (AU)

Objetivo: A prevalência da doença periodontal tem aumentado na maioria dos países, incluindo países em desenvolvimento e desenvolvidos, Afetando 20-50% da população global. Pacientes com Diabetes Mellitus tipo 2 (DM) com doença periodontal grave apresentaram risco 3,2 vezes maior de morte do que indivíduos sem periodontite. O objetivo deste estudo foi determinar a gravidade da periodontite utilizando o nível de IL-10 (Interleucina-10) no diabetes mellitus tipo 2. Materiais e Métodos: Este estudo transversalfoi realizadoseguindo as orientações e regulamentos do Comitê de Ética da Faculdade de Medicina Dentária da Universitas Airlangga. Noventa participantes,responderam um questionário e foram examinados , para o estado periodontal, baseadas no Índice Periodontal Comunitário (IPC) e medidas aleatórias de glicemia. Os dados do nível de IL-10 foram obtidos utilizando Fluido Crevicular Gengival (GCF). Resultados: Houve uma diferença significativa no estilo de vida em cada grupo. O nível mais alto de IL-10 foi encontrado no grupo com periodontite, seguido pela periodontite com o grupo DM tipo 2. Conclusão: O nível de IL-10 pode ser utilizado para determinar a gravidade da periodontite no DM tipo 2. A maioria dos participantes com maior nível de IL-10 estava no grupo periodontite seguida de periodontite com DM tipo 2. Altos níveis de IL-10 diminuiem a síntese do Fator de Necrose Tumoral Alfa (TNF-α), Interleucina-1 (IL-1), Interleucina-6 (IL-6), ativação de macrófagos e neutrófilos polimorfonucleares (PMN) (AU)

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells

Abstract Objective: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. Methodology: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. Results: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). Conclusions: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Clinical significance of serum microRNA-146a and inflammatory factors in children with Mycoplasma pneumoniae pneumonia after azithromycin treatment

Abstract Objective This study aimed to investigate the clinical significance of serum microRNA-146a and pro-inflammatory factors in children with Mycoplasma pneumoniae pneumonia after azithromycin treatment. microRNA-146a is known to regulate inflammatory responses, and excessive inflammation is a primary characteristic of MPP. Methods Children with MPP received conventional symptomatic therapy along with intravenous administration of azithromycin for one week. Serum levels of microRNA-146a and pro-inflammatory factors were measured using RT-qPCR and ELISA kits, respectively. The correlation between microRNA-146a and pro-inflammatory factors was analyzed by the Pearson method. Pulmonary function indexes were assessed using a pulmonary function analyzer, and their correlation with microRNA-146a and pro-inflammatory factors after treatment was evaluated. Children with MPP were divided into effective and ineffective treatment groups, and the clinical significance of microRNA-146a and pro-inflammatory factors was evaluated using receiver operating characteristic curves and logistic multivariate regression analysis. Results Serum microRNA-146a was downregulated in children with MPP but upregulated after azithromycin treatment, contrasting with the trend observed for pro-inflammatory factors. MicroRNA-146a showed a negative correlation with pro-inflammatory cytokines. Pulmonary function parameters were initially reduced in children with MPP, but increased after treatment, showing positive/inverse associations with microRNA-146a and pro-inflammatory factors. Higher microRNA-146a and lower pro-inflammatory factors predicted better efficacy of azithromycin treatment. MicroRNA-146a, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and forced expiratory volume in the first second/forced vital capacity (FEV1/FVC) were identified as independent factors influencing treatment efficacy. Conclusion Azithromycin treatment in children with MPP upregulates microRNA-146a, downregulates pro-inflammatory factors, and effectively improves pulmonary function.

Up-regulation of Proinflammatory Cytokines in Rostral Ventromedial Medulla Contributes to Chronic Postsurgical Pain by Promoting 5-HT Release / 中山大学学报(医学科学版)

ObjectiveTo investigate the role of proinflammatory cytokines tumor necrosis factor alpha （TNFα） and interleukin-1β （IL-1β） in rostral ventromedial medulla （RVM） in chronic postsurgical pain （CPSP） induced by skin/muscle incision and retraction （SMIR）. MethodsSD rats were randomly divided into 5 groups： ① Sham group； ② SMIR group； ③ SMIR+TNFα/IL-1β neutralizing antibody group； ④ SMIR+TNFα/IL-1β group and ⑤ SMIR+vehicle group. 50% paw mechanical withdrawal threshold （MWT） was measured by the up-down method， immunofluroscence was used to detect the TNFα and IL-1β expression and ELISA for the 5-Hydroxytryptamine （5-HT） level. ResultsSMIR elicited persistent nociceptive sensitization， upregulated TNFα and IL-1β expression in RVM neurons and astrocytes. Microinjection of TNFα or IL-1β neutralizing antibody into RVM inhibited the development of nociceptive sensitization and decreased the level of 5-HT in both RVM and spinal dorsal horn. While microinjection of recombinant TNFα or IL-1β into RVM enhanced the development of nociceptive sensitization and increased the level of 5-HT in both RVM and spinal dorsal horn. ConclusionUp-regulation of proinflammatory cytokines in RVM may contribute to SMIR induced CPSP by promoting 5-HT release.

Oncolytic Newcastle disease virus inhibits IL-6 induced migration and invasion of human glioblastoma U87MG cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨溶瘤新城疫病毒（NDV）对IL-6诱导的人胶质母细胞瘤U87MG细胞增殖、迁移和侵袭的作用及其可能的机制。方法：将U87MG细胞分为对照组、IL-6组、NDV组、NDV+IL-6组，其中IL-6组与NDV+IL-6组用75 ng/mL IL-6预处理1 h，其余组用DMEM预处理1 h，后分别用DMEM、75 ng/mL IL-6、1 HU NDV、1 HU NDV+75 ng/mL IL-6处理24 h。MTT法、细胞划痕实验和Transwell侵袭实验分别检测IL-6、NDV对U87MG细胞增殖、迁移和侵袭的影响，WB法检测各组细胞JAK2、p-JAK2、STAT3、p-STAT3和MMP2蛋白的表达水平。结果：与对照组相比，IL-6组细胞迁移率显著升高（P<0.05），侵袭细胞数目显著增多（P<0.01）；与IL-6组相比，NDV+IL-6组U87MG细胞增殖率显著降低（P<0.05），细胞迁移率和侵袭细胞数目均显著降低（均P<0.01）。WB实验结果显示，与对照组相比，IL-6组p-STAT3/STAT3比值显著升高（P<0.01），NDV组p-JAK2/JAK2、p-STAT3/STAT3比值显著降低（P<0.05，P<0.01），MMP-2蛋白表达量显著降低（P<0.01）；与IL-6组相比，NDV+IL-6组p-STAT3/STAT3比值、MMP-2蛋白表达量均显著降低（均P<0.05）。结论：NDV能抑制IL-6对人脑胶质瘤U87MG细胞迁移和侵袭的诱导作用，其机制可能与JAK2/STAT3信号通路的参与调控有关。

Role of IL-22/IL-22RA1 pathway in the malignant progression of oral squamous carcinoma and its mechanism / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨IL-22/IL-22受体A1（IL-22RA1）通路在口腔鳞状细胞癌（OSCC）恶性进展中的作用及其机制。方法：利用GEO数据库和免疫组化法分析IL-22RA1在OSCC组织及配对癌旁组织中的表达水平，采用组织芯片免疫组化法检测并分析IL-22RA1表达水平与OSCC患者临床病理特征的关系，通过EBI ArrayExpress数据库分析IL-22RA1表达水平与患者预后的关系。采用免疫荧光法检测IL-22和IL-22RA1在OSCC组织中表达水平并分析两者间的相关性。用RNA干扰技术敲减OSCC细胞WSU-HN4和CAL27的IL-22RA1表达，用qPCR法验证敲减效率。采用克隆形成实验、Transwell小室法分别检测IL-22对阴性对照（siNC）组和IL-22RA1敲减（siIL-22RA1）组OSCC细胞克隆形成及迁移能力的影响，WB法检测IL-22对OSCC细胞IL-22RA1表达水平及STAT1、STAT3和ERK1/2磷酸化水平的影响。结果：OSCC组织中IL-22RA1 mRNA的表达水平显著高于癌旁组织（P<0.05）。IL-22RA1表达水平与OSCC患者的肿瘤大小（P<0.05）、淋巴结转移（P<0.01）及预后不良（P<0.05）有关联。OSCC组织中的IL-22和IL-22RA1表达水平无明显关联，IL-22对OSCC细胞IL-22RA1表达无影响（均P>0.05）。IL-22可显著增强OSCC细胞的克隆形成和迁移能力（均P<0.01），并可激活OSCC细胞的STAT1、STAT3及ERK1/2信号分子；敲减OSCC细胞的IL-22RA1后，IL-22则无法发挥上述作用。结论：IL-22/IL-22RA1可通过调控细胞增殖和迁移促进OSCC的生长和转移，其下游机制可能是激活ERK1/2-STAT1/3信号通路。

Improvement effect and mechanism of total flavonoids from Rosa multiflora root on vascular injury in rheumatoid arthritis model rats / 中国药房

OBJECTIVE To study the improvement effect of total flavonoids from Rosa multiflora root on vascular injury in rheumatoid arthritis （RA） model rats and its potential mechanism. METHODS Female Wistar rats were randomly divided into normal control group， model group， aspirin group （positive control， 30 mg/kg）， low-dose and high-dose groups of total flavonoids from R. multiflora root （4.15， 8.30 g/kg， by crude drug）， with 10 rats in each group. Except for the normal control group， the RA model was induced in other groups by collagen induction and high-fat diet. After 14 days of modeling， they were given corresponding drug solution/0.5% sodium carboxymethyl cellulose solution intragastrically， once a day， for 36 consecutive days. The total body score， arthritis index （AI） and swollen joint count （SJC） of the rats were evaluated regularly. After the last medication， serum levels of interleukin-6 （IL-6）， intercellular adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule- 1 （VCAM-1） were determined. The pathological morphological changes in the vascular tissue of thoracic aorta were observed； the protein expression of Toll-like receptor 4 （TLR4） and the protein phosphorylation levels of Janus kinase 2 （JAK2） and signal transduction and activator of transcription 3 （STAT3） in vascular tissue of thoracic aorta were measured. RESULTS Compared with the normal control group， serum levels of IL-6， ICAM-1 and VCAM-1， protein expression of TLR4， and the protein phosphorylation levels of JAK2 and STAT3 in vascular tissue of thoracic aorta were increased significantly in model group （P＜ 0.01）. The atherosclerotic plaque （atheroma）， cholesterol crystal， lymphocyte infiltration and a small number of unbroken foam cell aggregation could be seen in the vascular tissue of thoracic aorta. Compared with the model group， total body score （except for the low-dose group）， AI and SJC were decreased significantly in groups of total flavonoids from R. multiflora root on the 28th day （P＜0.05 or P＜0.01）； total body score，AI and SJC were decreased significantly in low-dose group of total flavonoids from R. multiflora root on the 49th day （P＜0.05 or P＜0.01）； the other quantitative indicators in serum and vascular tissue were significantly reversed in groups of total flavonoids from R. multiflora root （P＜0.05 or P＜0.01）， and pathological damage of vascular tissue was significantly relieved. CONCLUSIONS Total flavonoids from R. multiflora root can significantly improve vascular injury in RA model rats， and its mechanism may be related to reducing the protein expression of TLR4 in vascular tissue and inhibiting the activation of IL-6/JAK2/ STAT3 signaling pathway.

Effects of gastrodin on the expression of BDNF and IL-6 in the striatum of rats with cerebral ischemia / 中国临床药理学与治疗学

AIM: To investigate the effect of gastrodin on the expression of brain-derived neurotrophic factor (BDNF) and interleukin-6 (IL-6) in the striatum of cerebral ischemia rats, and to explore the potential mechanism of gastrodin in treating cerebral ischemia. METHODS: The rats were randomly divided into four groups: normal, sham, model, and gastrodin groups, each consisting of 10 rats. After successful modeling using middle cerebral artery occlusion (MCAO), the gastrodin group received intraperitoneal injection of gastrodin injection at a dose of 10 mg/kg once a day for 14 consecutive days. Pathological changes in striatal neurons were observed using Nissl staining. Immunohistochemistry was utilized to detect positive expression of BDNF and IL-6 proteins in the striatum. Additionally, immunoblot analysis was performed to determine the expression levels of BDNF and IL-6 proteins in the striatum. RESULTS: Nissl staining revealed clear and intact structures of striatal neurons in the normal and sham groups, with tightly arranged cells. In the model group, the number of cells was significantly reduced compared to the sham group (P0.05). Compared to the sham group, the model group showed a decrease in the protein expression level of BDNF in the striatum on the ischemic side (P<0.01) and an increase in the protein expression level of IL-6 (P<0.05, P<0.01). In contrast, the gastrodin group showed an increase in the protein expression level of BDNF in the striatum on the ischemic side (P<0.05, P<0.01) and a decrease in the protein expression level of IL-6 (P< 0.05, P<0.01) compared to the model group. CONCLUSION: Gastrodin has a significant protective effect on striatal injury caused by cerebral ischemia, and its mechanism may be related to the up-regulation of the anti-inflammatory factor BDNF and the down-regulation of the pro-inflammatory factor IL-6.

Mechanism of monoclonal antibody against interleukin IL-17A regulating autophagy and inflammation in gout / 中国生物制品学杂志

@#Objective To investigate the mechanism of anti-IL-17A monoclonal antibody(secukinumab) regulating autophagy and inflammation in gout.Methods The peripheral venous blood samples from 57 patients with acute gout(AG),57patients with intermittent gout(IG) and 82 healthy volunteers were collected and measured for the mRNA transcription levels of autophagy-related genes(ATGs) ATG4B,ATG7, A TG16L1,Beclin-1 and LC3B by RT-qPCR.The model of AG inflammation was established by adding monosodium urate(MSU) crystals into the peripheral venous blood samples of healthy volunteers,and the transcription and protein expression of IL-1β were detected by RT-qPCR and ELISA at 0,1,2,4,6 and8 h and different concentrations(0,100,200 and 400 μmol/L) of secukinumab.The peripheral blood samples of healthy volunteers were divided into control(without MSU treatment),MSU(100 μg/mL),MSU+colchicine(100 μg/mL+30 μg/mL) and MSU+secukinumab(100 μg/mL+400 μmol/L) groups,which were detected for the mRNA transcription and protein expression of IL-1 β and ATGs by RT-qPCR and Western blot,and for the expression of IL-1β,IL-12 and IL-35 by ELISA.Results The mRNA expression levels of ATG4B, Beclin-1 and LC3B in AG,IG and healthy control groups were significantly different(F=3.896,11.78 and 3.856,respectively,each P <0.05),among which the mRNA levels in AG were lower than those in IG and HC groups(t=2.692,3.234,2.231 and 2.085,4.795,2.748,respectively,each P <0.05);the expression levels of ATG16L1 mRNA were significantly different in the three groups(F=7.949,P <0.001),and was significantly lower in AG group than HC group(t=3.860,P <0.001).In AG inflammation model,the mRNA and protein expression of IL-1 β reached their peak in 2—4 h,and the anti-inflammation effect of secukinumab was the strongest at the concentration of 400 μmol/L.Compared with MSU group,the mRNA levels of ATG16L1 and LC3B(t=2.343 and 2.916,respectively,each P <0.05) as well as the expression levels of ATG4B,ATG7,Beclin-1,ATG16L1 and LC3B-Ⅱ proteins(t=28.84,11.6,8.402,4.124 and 2.458,respectively,each P <0.05) in MSU+secukinumab group decreased significantly.The expression levels of IL-12 and IL-35 in the control,MSU,MSU+colchicine and MSU+secukinumab groups showed significant difference(F=7.009 and 6.518,respectively,each P <0.01).Compared with MSU group,the expression level of IL-12 significantly decreased(t=2.604,P <0.05)in MSU+secukinumab group,and the expression level of IL-35 also decreased,while with no significant difference(t=1.928,P> 0.05).Conclusion Secukinumab can regulate the mRNA and protein expression of ATGs,reduce the levels of pro-inflammatory cytokines,and inhibit gout inflammation,which provides a reference for the treatment of gout.

Construction of human interleukin-26 lentivirus expression plasmid and gene knockout plasmid and their activity verification in HEK293T cells / 中国生物制品学杂志

@#Objective To construct a lentivirus-based expression plasmid and gene knockout plasmid of human interleukin(IL)-26 so as to lay a foundation of studying the function of IL-26 gene in cell signaling pathway and autophagy.Methods IL-26 gene sequence was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pCDH-CMVMCS-EF1-copGFP eukaryotic expression vector to construct overexpression plasmid;Four knockout targets,Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sgRNA2,were designed based on the exon sequence of IL-26,and constructed into lentiCRISPRv2 vector by CRISPR/Cas9 technology to construct gene knockout plasmid. The overexpression plasmid and gene knockout plasmid were transiently transfected into HEK293T cells respectively,and the expression of IL-26 was verified by RT-qPCR and Western blot. In addition,amino acid sequence analysis,structure prediction and subcellular localization observation of IL-26 were performed.Results The results of restriction digestion,sequencing and bioinformatics analysis showed that IL-26 was 516 bp in length,encoding 171 amino acids. The IL-26 mRNA level and protein level of HEK293T cells transfected with IL-26 overexpression plasmid increased by 656. 789 times and 1. 978 times respectively with significant differences as compared with the normal control group(t = 17. 976 and 7. 859,P < 0. 000 1 and < 0. 001,respectively). With the transfection of 4 knockout targets Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sg-RNA2 into HEK293T cells,the expression of IL-26 decreased by 0. 930,0. 980,0. 523 3 and 0. 316 9 times,respectively,among which Exon3sgRNA2 significantly down-regulated the expression of IL-26(t = 7. 440,P < 0. 001). IL-26protein showed signal peptide structure and certain transmembrane function in the first 22 amino acids,which existed in cytoplasm.Conclusion IL-26overexpression and gene knockout plasmids were successfully constructed,which laid a foundation of the follow-up study of the function of IL-26.

Immunostimulatory gene therapy combined with checkpoint blockade reshapes tumor microenvironment and enhances ovarian cancer immunotherapy

Immune evasion has made ovarian cancer notorious for its refractory features, making the development of immunotherapy highly appealing to ovarian cancer treatment. The immune-stimulating cytokine IL-12 exhibits excellent antitumor activities. However, IL-12 can induce IFN-γ release and subsequently upregulate PDL-1 expression on tumor cells. Therefore, the tumor-targeting folate-modified delivery system F-DPC is constructed for concurrent delivery of IL-12 encoding gene and small molecular PDL-1 inhibitor (iPDL-1) to reduce immune escape and boost anti-tumor immunity. The physicochemical characteristics, gene transfection efficiency of the F-DPC nanoparticles in ovarian cancer cells are analyzed. The immune-modulation effects of combination therapy on different immune cells are also studied. Results show that compared with non-folate-modified vector, folate-modified F-DPC can improve the targeting of ovarian cancer and enhance the transfection efficiency of pIL-12. The underlying anti-tumor mechanisms include the regulation of T cells proliferation and activation, NK activation, macrophage polarization and DC maturation. The F-DPC/pIL-12/iPDL-1 complexes have shown outstanding antitumor effects and low toxicity in peritoneal model of ovarian cancer in mice. Taken together, our work provides new insights into ovarian cancer immunotherapy. Novel F-DPC/pIL-12/iPDL-1 complexes are revealed to exert prominent anti-tumor effect by modulating tumor immune microenvironment and preventing immune escape and might be a promising treatment option for ovarian cancer treatment.

Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Interleukin-37 has A Promising Cardioprotective Effect on Isoproterenol-Induced Myocardial Infarction / La Interleucina-37 tiene un Efecto Cardioprotector Prometedor sobre el Infarto de Miocardio Inducido por Isoproterenol

SUMMARY: The main cause of mortality and disability globally is myocardial infarction (MI). Isoproterenol (ISO), a β-adrenoceptor agonist, has been used to induce rat myocardial necrosis. Whereas interleukin-37 (IL-37) has anti-inflammatory and cytoprotective properties. The study aimed to investigate the potential protective effects of IL-37 administration on cardiac architecture, oxidative stress, and inflammatory markers during ISO-induced MI in rats. Three groups of adult male rats were used in this study, the normal control group (n=8), ISO-induced MI group (n=8) that received isoproterenol hydrochloride (ISO) (100 mg/kg/day, SC, for the first 2 consecutive days), and IL-37-treated group (ISO+IL-37) (n=8) that received recombinant human IL-37 (40 µg/kg /day, intraperitoneally, for 2 weeks during and after ISO injections. Heart rate (HR.) and ECG changes were monitored. Some oxidative stress markers such as superoxide dismutase (SOD), nitric oxide (NOx), malondialdehyde (MDA), and glutathione (GSH) tissue levels in the tissue homogenate were assayed. Interleukin- 6 (IL-6), tumor necrosis factor- α (TNF-α), caspase-8, P53, and C- reactive protein (CRP) were among the inflammatory markers examined. In addition, serum levels of creatinine kinase (CK-MB) and lactate dehydrogenase (LDH) were analyzed to evaluate the myocardial injury. For histological analysis, tissues were sectioned, fixed in paraffin, and stained with hematoxylin and eosin (H&E), Masson Trichrome and, immunohistochemical against NF-kB, TNF-α, and Caspase-9. IL-37 improved ECG changes, cardiac enzyme markers, and some inflammatory markers of oxidative stress in ISO-induced MI. It also improved the histopathological and immunohistochemical changes in MI. In conclusion: IL-37 might be a promising therapeutic modality in myocardial infarction.

La principal causa de mortalidad y discapacidad a nivel mundial es el infarto de miocardio (IM). El isoproterenol (ISO), un agonista de los receptores adrenérgicos β, se ha utilizado para inducir necrosis miocárdica en ratas. Mientras que la interleucina-37 (IL-37) tiene propiedades antiinflamatorias y citoprotectoras. El estudio tuvo como objetivo investigar los posibles efectos protectores de la administración de IL-37 en la arquitectura cardíaca, el estrés oxidativo y los marcadores inflamatorios durante el infarto de miocardio inducido por ISO en ratas. En este estudio se utilizaron tres grupos de ratas macho adultas, el grupo control normal (n=8), el grupo con IM inducido por ISO (n=8) que recibió clorhidrato de isoproterenol (ISO) (100 mg/kg/día, SC, durante los primeros 2 días consecutivos) y el grupo tratado con IL-37 (ISO+IL- 37) (n=8) que recibió IL-37 humana recombinante (40 µg/kg/día, por vía intraperitoneal, durante 2 semanas durante y después de las inyecciones de ISO. Se monitorearon la frecuencia cardíaca (FC) y los cambios en el ECG. Se analizaron algunos marcadores de estrés oxidativo como la superóxido dismutasa (SOD), el óxido nítrico (NOx), el malondialdehído (MDA) y los niveles tisulares de glutatión (GSH) en el homogeneizado de tejido. La interleucina-6 (IL-6), el factor de necrosis tumoral-α (TNF-α), la caspasa-8, la P53 y la proteína C reactiva (CRP) se encontraban entre los marcadores inflamatorios examinados. Se analizaron los niveles de creatinoquinasa (CK-MB) y lactato deshidrogenasa (LDH) para evaluar la lesión miocárdica; para el análisis histológico se seccionaron los tejidos, se fijaron en parafina y se tiñeron con hematoxilina y eosina (H&E), Tricromo de Masson e inmunohistoquímica contra NF-kB, TNF-α y Caspasa-9. IL-37 mejoró los cambios de ECG, los marcadores de enzimas cardíacas y algunos marcadores inflamatorios de estrés oxidativo en el IM inducido por ISO. Además mejoró los cambios histopatológicos e inmunohistoquímicos en MI. En conclusión: la IL-37 podría ser una modalidad terapéutica prometedora en el infarto de miocardio.

Evaluation of IL-4, MIP-1α, and MMP-9 gene expression levels in peri-implant tissues in peri-implantitis

Abstract This case-control study evaluated the gene expression levels of interleukin (IL)-4, macrophage inflammatory protein type 1 alpha (MIP-1α), and metalloproteinase (MMP)-9, factors involved in the formation of giant cells in healthy peri-implant tissue and peri-implantitis. Thirty-five subjects (15 healthy and 20 with peri-implantitis), who met the inclusion and exclusion criteria, were included in this study. The peri-implant tissue biopsies were subjected to total RNA extraction, DNAse treatment, and cDNA synthesis. Subsequently, the reaction of real-time PCR was performed to evaluate the gene expression levels of IL-4, MIP-1α, and MMP-9 concerning the reference gene. IL-4 gene expression showed higher (18-fold) values in the Peri-Implantitis Group of Patients when compared with the Healthy (Control) Group (p<0.0001). Although MIP- 1α and MMP-9 gene expression levels were higher in diseased implants, they showed no significant differences (p=0.06 and p=0.2337), respectively. Within the limitations of this study, the results showed that in tissues affected by peri-implantitis, only levels of Il-4 were increased when compared with tissues in the control group.

Resumo Este estudo caso-controle teve como objetivo avaliar a expressão gênica dos níveis de interleucina (IL)-4, proteína inflamatória de macrófagos tipo alfa 1 (MIP-1α) e metalopreoteinase (MMP)-9, todos fatores envolvidos na formação de células gigantes em tecidos peri-implantares saudáveis e com peri-implantite. Trinta e cinco indivíduos (15 saudáveis e 20 com peri-implantite) foram incluídos nesse estudo seguindo os critérios de inclusão e exclusão. Os tecidos peri-implantares foram submetidos a extração do RNA total, tratamento de DNAse e síntese de cDNA. Subsequentemente, a reação de PCR em tempo real foi realizada para avaliar os níveis da expressão de IL-4, MIP-1α, e MMP-9 em relação ao gene de referência. O nível de expressão de IL-4 foi estatisticamwente maior (18 vezes) nos tecidos de pacientes com peri-implantite quando comparados aos pacientes saudáveis (grupo controle) (p<0,0001). Embora os níveis de expressão de MIP- 1α e MMP-9 apresentassem maiores valores nos implantes doentes, esses níveis não foram estatisticamente significantes (p=0.06 and p=0.2337) respectivamente. Dentro das limitações desse estudo, os resultados mostraram que nos tecidos afetados pela peri-implantite, apenas os nívies de IL-4 estavam aumentados quando comparados ao grupo controle.

Correlation of IL36RN and CARD14 mutations with clinical manifestations and laboratory findings in patients with generalised pustular psoriasis

Background: Generalized pustular psoriasis (GPP) is a chronic disease associated with genetic factors related to mutations of the interleukin 36 receptor antagonist gene (IL36RN) and the caspase recruitment domain 14 gene (CARD14). However, the relevance of these mutations to the clinical features and severity of GPP remains unclear. Aims: Our objective was to correlate the presence of IL36RN and CARD14 mutations with the clinical and laboratory findings in patients with GPP. Methods: This cross-sectional descriptive study was conducted in 64 subjects with GPP. Clinical manifestations were recorded and the severity was graded as mild, moderate, or severe. Routine laboratory tests were performed and blood samples were collected for Sanger sequencing. The clinical data of patients were compared among the different mutation groups. Results: The two main variants of IL36RN were c.115+6T > C (p.Arg10ArgfsX1) and c.227C > T (p.Pro76Leu). The major CARD14 mutations were c.2458C > T (p.Arg820Trp), c.1641C > T (p.Arg547Ser), and c.1753G > A transitions. Provocative factors were uncommon in the group with both IL36RN and CARD14 mutations. Drugs (unspecified), especially herbals, were the most common triggers. A history of psoriasis was frequent in patients with only CARD14 mutations, but fever was uncommon. The c.1641C > T mutation was associated with leukocytosis > 15000/mm3 and the c.1753G > A mutation was associated with hypoalbuminemia <3.8g/dL. Both the c.115+6T > C and c.227C > T variants of IL36RN were associated with fever ?38.5°C while the c.115+6T > C variant was also associated with geographic tongue. No gene mutations were associated with the total severity and severity grades. Limitations: Four patients without the two major IL36RN mutations were excluded from the study. Conclusion: The presence of IL36RN and CARD14 mutations were associated with a history of psoriasis, various provocative factors, fever, leukocytosis, hypoalbuminemia, and geographic tongue. Further studies to explore the role of these mutations in therapeutic efficacy and disease outcomes are necessary.

Utilización precoz de tocilizumab en pacientes internados con COVID-19 grave y crítica: un estudio multicéntrico en la Provincia de Buenos Aires / Early use of tocilizumab in hospitalized patients with severe and critical COVID-19 in the Province of Buenos Aires: a multicentric study

Resumen Introducción: El objetivo principal del estudio fue evaluar la mortalidad en los pacientes con COVID-19 graves y críticos, que recibieron tocilizumab (TCZ) -un antagonista monoclonal del receptor de IL-6- de forma temprana vs. tardía. Métodos: Cohorte retrospectiva multicéntrica de pacientes >18 años internados con COVID-19 desde el 1/7/2021-1/8/2022, con 5-7 puntos de gravedad inicial (GI) según Escala de la OMS. Se consideró adminis tración temprana o tardía a la infusión de TCZ ≤ ó > a 48 h del ingreso. Las variables de resultado fueron mortalidad a 28 días y cambio de la GI. Los factores relacionados con la mortalidad fueron evaluados con regresión de Cox. Resultados: Se incluyeron 266 pacientes, 159(60%) varones; edad 58(± 15); con hipertensión arterial (43%), obesidad (37%) y diabetes (27%);70 presentaban GI = 5 (oxígeno suplementario), 143 GI = 6 (ventilación no inva siva o cánula nasal de alto flujo) y 53 GI = 7 (ventilación mecánica invasiva). La mortalidad a 28 días fue 42%, asociada independientemente a: edad, obesidad, GI, días entre la internación y administración del TCZ, y días entre la fecha de inicio de síntomas y el TCZ. La mortalidad para GI 5, 6 y 7 fue 26%, 39% y 72%, respectivamente; 76% y 62% de los pacientes permanecieron estables o mejoraron la GI a los días 3 y 7 de la infusión de TCZ. La mortalidad a 28 días fue 39% (TCZ temprano) vs. 57% (TCZ tardío); p = 0.02; HR = 0.63[0.41-0.99, p = 0.05]). Discusión: Estos resultados apoyan la administración temprana de TCZ en pacientes con COVID-19 grave y crítica.

Abstract Introduction: Tocilizumab (TCZ), an IL-6 receptor antagonist monoclonal antibody is warranted in severe and critically-ill COVID-19 patients. The objective was to evaluate 28-day mortality of patients with severe or critical COVID-19 treated with early vs delayed TCZ. Methods: Multicenter, retrospective cohort study in cluding patients>18 years hospitalized between 7/1/2021- 8/1/2022 with confirmed COVID-19, with 5, 6 and 7 points of WHO Ordinal Initial Severity Scale [SS]. Early or late administration was considered if TCZ was administered before or after 48 hours from admission. Outcomes were28-day mortality and change of SS. Factors related to 28-day mortality were evaluated with Cox regression. Results: 266 patients were included, 159(60%) male; aged 58(± 15); frequent comorbidities were hypertension (42%), obesity (37%) and diabetes (27%). Seventy patients had a SS = 5 (Supplemental O2), 143 had SS = 6 (NIV/ HFNC), and 53 had SS = 7 (IMV). 28-day mortality was 42%(112/266); predictors were age, obesity, higher SS, days between hospitalization and TCZ administration, and fewer days between symptoms onset and TCZ. Mortality of SS 5, 6 and 7 was 26%, 39% and 72% respectively. Com pared with baseline SS points, 76% and 62% of patients remained stable or improved on days 3 and 7 since TCZ administration. 28-day mortality was lower when TCZ was administered before 48 hours (39% vs 57%; p = 0.02; HR = 0.63;[0.41-0.99, p = 0.05]). Discussion: This study supports the early use of TCZ in patients with severe or critical COVID-19.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

A Rat Model of Rheumatoid Arthritis: TDZD-8 is Associated with the Protection Against the Induction of the Synovium Knee Joint IL-17A/GSK3β/ROS/α-SMA Axis of Fibrosis

SUMMARY: Rheumatoid arthritis (RA) that affects the synovial knee joint causes swelling of the synovial membrane and tissue damage. Interleukin-17A (IL-17A) and the enzyme glycogen synthase kinase-3β (GSK3β) are involved in the pathogenesis of RA. The link between IL-17A, GSK3β, the oxidative stress, and the profibrogenic marker alpha-smooth muscle actin (α-SMA) with and without TDZD-8, GSK3β inhibitor has not been studied before. Consequently, active immunization of rats was performed to induce RA after three weeks using collagen type II (COII) injections. The treated group received daily injection of 1 mg/kg TDZD-8 for 21 days following the immunization protocol (COII+TDZD-8). Blood and synovium tissue samples were harvested at the end of the experiment. RA development was confirmed as corroborated by a substantial increase in blood levels of the highly specific autoantibody for RA, anti-citrullinated protein antibody as well as augmentation of reactive oxidative species (ROS) levels measured as lipid peroxidation. RA induction also increased synovium tissue levels of IL-17A and the profibrogenic marker, α-SMA. All these parameters seemed to be significantly (p<0.0001) ameliorated by TDZD-8. Additionally, a significant correlation between IL-17A, ROS, and α-SMA and biomarkers of RA was observed. Thus, knee joint synovium RA induction augmented IL-17A/GSK3β/ROS/α-SMA axis mediated arthritis in a rat model of RA, which was inhibited by TDZD-8.

La artritis reumatoide (AR) que afecta la articulación sinovial de la rodilla provoca inflamación de la membrana sinovial y daño tisular. La interleucina-17A (IL-17A) y la enzima glucógeno sintasa quinasa-3β (GSK3β) están involucradas en la patogenia de la AR. No se ha estudiadol vínculo entre IL-17A, GSK3β, el estrés oxidativo y el marcador profibrogénico actina de músculo liso alfa (α-SMA) con y sin inhibidor de TDZD-8, GSK3β. En consecuencia, se realizó una inmunización activa de ratas para inducir la AR después de tres semanas usando inyecciones de colágeno tipo II (COII). El grupo tratado recibió una inyección diaria de 1 µg/ kg de TDZD-8 durante 21 días siguiendo el protocolo de inmunización (COII+TDZD-8). Se recogieron muestras de sangre y tejido sinovial al final del experimento. El desarrollo de AR se confirmó como lo corroboró el aumento sustancial en los niveles sanguíneos del autoanticuerpo altamente específico para AR, el anticuerpo antiproteína citrulinada, así como el aumento de los niveles de especies oxidativas reactivas (ROS) medidos como peroxidación lipídica. La inducción de AR también aumentó los niveles de tejido sinovial de IL-17A y el marcador profibrogénico, α-SMA. Todos estos parámetros parecían mejorar significativamente (p<0,0001) con TDZD-8. Además, se observó una correlación significativa entre IL- 17A, ROS y α-SMA y biomarcadores de AR. Por lo tanto, la inducción de AR en la sinovial de la articulación de la rodilla aumentó la artritis mediada por el eje IL-17A/GSK3β/ROS/α-SMA en un modelo de rata de AR, que fue inhibida por TDZD-8.

The role of pro-inflammatory cytokines – tumor necrosis factor-alpha and interleukin 6 – in the evolution of diabetic nephropathy: A comparative study

Background: Diabetes is highly prevalent and it is responsible for the increased financial burden on healthcare. Type II diabetes is a more prevalent form of diabetes. Uncontrolled and unsupervised type II diabetes may lead to various microvascular and macrovascular complications which are responsible for high morbidity and mortality. Diabetic nephropathy (DN) is a common complication characterized by the expansion of mesangial cells with thickening of the basement and nodular glomerulosis. TNF-alpha and IL-6 play an important role in causing detrimental changes leading to nephropathy. The study of the role of these inflammatory cytokines in patients with DN may help in the early diagnosis and management. Aims and Objectives: The objectives of this study were to compare the levels of proinflammatory cytokines, TNF-?, and IL-6 in the evolution of DN patients. Materials and Methods: The present study was conducted in the Department of Biochemistry, in collaboration with the Department of Medicine (Nephrology unit); Pt. B.D. Sharma, Post Graduate Institute of Medical Sciences, Rohtak after ethical clearance. Forty patients with DN (Stages 3, 4, and 5) and forty patients with diabetes mellitus without nephropathy were taken up for study after taking informed consent. Results: The mean serum TNF-? levels in cases was 33.05 ± 29.22 pg/mL and in controls was 17.67 ± 12.33 pg/mL. On the basis of unpaired t-test, the difference between the groups was statistically highly significant (P < 0.05). The mean serum interleukin-6 levels in cases was 24.92 ± 30.16 pg/mL (2.95–155.55 pg/mL) and in controls was 6.76 ± 5.82 pg/mL (2.22–35.42 pg/mL). On the basis of the t-test, the difference between the groups was statistically highly significant (P < 0.05). Conclusion: TNF-? and IL-6 may serve as potential biomarkers for patients with DN and also in the development of newer therapeutic modalities for the prevention and treatment of DN.