Investigation on the growth factor regulatory network of dermal fibroblasts in mouse full-thickness skin defect wounds based on single-cell RNA sequencing / 中华烧伤杂志

Objective: To explore the heterogeneity and growth factor regulatory network of dermal fibroblasts (dFbs) in mouse full-thickness skin defect wounds based on single-cell RNA sequencing. Methods: The experimental research methods were adopted. The normal skin tissue from 5 healthy 8-week-old male C57BL/6 mice (the same mouse age, sex, and strain below) was harvested, and the wound tissue of another 5 mice with full-thickness skin defect on the back was harvested on post injury day (PID) 7. The cell suspension was obtained by digesting the tissue with collagenase D and DNase Ⅰ, sequencing library was constructed using 10x Genomics platform, and single-cell RNA sequencing was performed by Illumina Novaseq6000 sequencer. The gene expression matrices of cells in the two kinds of tissue were obtained by analysis of Seurat 3.0 program of software R4.1.1, and two-dimensional tSNE plots classified by cell group, cell source, and gene labeling of major cells in skin were used for visual display. According to the existing literature and the CellMarker database searching, the expression of marker genes in the gene expression matrices of cells in the two kinds of tissue was analyzed, and each cell group was numbered and defined. The gene expression matrices and cell clustering information were introduced into CellChat 1.1.3 program of software R4.1.1 to analyze the intercellular communication in the two kinds of tissue and the intercellular communication involving vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) signal pathways in the wound tissue, the relative contribution of each pair of FGF subtypes and FGF receptor (FGFR) subtypes (hereinafter referred to as FGF ligand receptor pairs) to FGF signal network in the two kinds of tissue, and the intercellular communication in the signal pathway of FGF ligand receptor pairs with the top 2 relative contributions in the two kinds of tissue. The normal skin tissue from one healthy mouse was harvested, and the wound tissue of one mouse with full-thickness skin defect on the back was harvested on PID 7. The multiple immunofluorescence staining was performed to detect the expression and distribution of FGF7 protein and its co-localized expression with dipeptidyl peptidase 4 (DPP4), stem cell antigen 1 (SCA1), smooth muscle actin (SMA), and PDGF receptor α (PDGFRα) protein. Results: Both the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7 contained 25 cell groups, but the numbers of cells in each cell group between the two kinds of tissue were different. Genes PDGFRα, platelet endothelial cell adhesion molecule 1, lymphatic endothelial hyaluronic acid receptor 1, receptor protein tyrosine phosphatase C, keratin 10, and keratin 79 all had distinct distributions on two-dimensional tSNE plots, indicating specific cell groups respectively. The 25 cell groups were numbered by C0-C24 and divided into 9 dFb subgroups and 16 non-dFb groups. dFb subgroups included C0 as interstitial progenitor cells, C5 as adipose precursor cells, and C13 as contractile muscle cells related fibroblasts, etc. Non-dFb group included C3 as neutrophils, C8 as T cells, and C18 as erythrocytes, etc. Compared with that of the normal skin tissue of healthy mice, the intercellular communication in the wound tissue of full-thickness skin defected mice on PID 7 was more and denser, and the top 3 cell groups in intercellular communication intensity were dFb subgroups C0, C1, and C2, of which all communicated with other cell groups in the wound tissue. In the wound tissue of full-thickness skin defected mice on PID 7, VEGF signals were mainly sent by the dFb subgroup C0 and received by vascular related cell groups C19 and C21, PDGF signals were mainly sent by peripheral cells C14 and received by multiple dFb subgroups, EGF signals were mainly sent by keratinocyte subgroups C9 and C11 and received by the dFb subgroup C0, and the main sender and receiver of FGF signals were the dFb subgroup C6. In the relative contribution rank of FGF ligand receptor pairs to FGF signal network in the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7, FGF7-FGFR1 was the top 1, and FGF7-FGFR2 or FGF10-FGFR1 was in the second place, respectively; compared with those in the normal skin tissue, there was more intercellular communication in FGF7-FGFR1 signal pathway, while the intercellular communication in FGF7-FGFR2 and FGF10-FGFR1 signal pathways decreased slightly or did not change significantly in the wound tissue; the intercellular communication in FGF7-FGFR1 signal pathway in the wound tissue was stronger than that in FGF7-FGFR2 or FGF10-FGFR1 signal pathway; in the two kinds of tissue, FGF7 signal was mainly sent by dFb subgroups C0, C1, and C2, and received by dFb subgroups C6 and C7. Compared with that in the normal skin tissue of healthy mouse, the expression of FGF7 protein was higher in the wound tissue of full-thickness skin defected mouse on PID 7; in the normal skin tissue, FGF7 protein was mainly expressed in the skin interstitium and also expressed in the white adipose tissue near the dermis layer; in the two kinds of tissue, FGF7 protein was co-localized with DPP4 and SCA1 proteins and expressed in the skin interstitium, co-localized with PDGFRα protein and expressed in dFbs, but was not co-localized with SMA protein, with more co-localized expression of FGF7 in the wound tissue than that in the normal skin tissue. Conclusions: In the process of wound healing of mouse full-thickness skin defect wound, dFbs are highly heterogeneous, act as potential major secretory or receiving cell populations of a variety of growth factors, and have a close and complex relationship with the growth factor signal pathways. FGF7-FGFR1 signal pathway is the main FGF signal pathway in the process of wound healing, which targets and regulates multiple dFb subgroups.

Dramatic clinical response to dabrafenib plus trametinib in anaplastic thyroid carcinoma and the challenges faced during the COVID-19 pandemic

SUMMARY Anaplastic thyroid carcinoma is the rarest tumor of the thyroid gland, representing less than 2% of clinically recognized thyroid cancers. Typically, it has an extremely rapid onset, fatal outcomes in most cases, and a median overall survival of 3 to 10 months despite aggressive multidisciplinary management. The presence of targetable mutations in anaplastic thyroid carcinoma patients is an opportunity for treatment when conventional therapeutics approaches are not effective, a frequent situation in the majority of patients. We present our experience in the management of a patient with unresectable anaplastic thyroid cancer who had a remarkable and rapid response to treatment with dabrafenib and trametinib during the COVID-19 pandemic. After four weeks of dabrafenib 150 mg twice daily plus trametinib 2 mg daily, he showed a dramatic reduction of the cervical mass around 90%. Nearly eight weeks under treatment with dabrafenib plus trametinib, the patient remains with minimal locoregional disease without distant metastases.

Optimization of a cucurbit6uril-based real-time label-free method for analyzing the activity of ornithine decarboxylase / 生物工程学报

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.

Olmesartan-associated Enteropathy / 中国医学科学院学报

Olmesartan,an angiotensin Ⅱ receptor blocker,is a commonly used antihypertensive drug.Several case reports and cohort studies in recent years have described a severe gastrointestinal adverse event with chronic diarrhea,intestinal malabsorption,and weight loss after the administration of olmesartan.In such cases,the patients recovered after discontinuing olmesartan.This adverse effect is called olmesartan-associated enteropathy(OAE).This article reviews the potential pathogenesis and clinical characteristics of OAE,which broadens the disease spectrum for the differential diagnosis of chronic diarrhea and intestinal malabsorption.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M

Azeliragon ameliorates Alzheimer's disease via the Janus tyrosine kinase and signal transducer and activator of transcription signaling pathway

OBJECTIVES: TTP488, an antagonist of the receptor for advanced glycation end-products, was evaluated as a potential treatment for patients with mild-to-moderate Alzheimer's disease (AD). However, the mechanism underlying the protective action of TTP488 against AD has not yet been fully explored. METHODS: Healthy male rats were exposed to aberrant amyloid β (Aβ) 1-42. Lipopolysaccharide (LPS) and the NOD-like receptor family pyrin domain containing 1 (NLRP1) overexpression lentivirus were injected to activate the NLRP1 inflammasome and exacerbate AD. TTP488 was administered to reverse AD injury. Finally, tofacitinib and fludarabine were used to inhibit the activity of Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) to prove the relationship between the JAK/STAT signaling pathway and TTP488. RESULTS: LPS and NLRP1 overexpression significantly increased the NLRP1 levels, reduced neurological function, and aggravated neuronal damage, as demonstrated by the impact latency time of, time spent by, and length of the platform covered by, the mice in the Morris water maze assay, Nissl staining, and immunofluorescence staining in rats with AD. CONCLUSIONS: TTP488 administration successfully reduced AD injury and reversed the aforementioned processes. Additionally, tofacitinib and fludarabine administration could further reverse AD injury after the TTP488 intervention. These results suggest a new potential mechanism underlying the TTP488-mediated alleviation of AD injury.

Trametinib and dabrafenib induced rhabdomyolysis, renal failure, and visual loss. Report of one case

ABSTRACT MEK- and BRAF-inhibitors trametinib and dabrafenib are successfully used for BRAF-mutated, metastasizing melanoma, but these compounds may induce side effects. We report a 50 years old female with BRAF-mutated metastasizing melanoma who received trametinib (2 mg/d) and dabrafenib (200 mg/d) after using interferon without benefit. Shortly after starting trametinib/dabrafenib, she experienced an inability to abduct the left eye. Eight days after starting this therapy the patient experienced loss of appetite, vomiting, diarrhea, vertigo, and fever of 40°C. Two days later she experienced visual loss, requiring permanent support for her daily activities. Two further days later myoglobinuria appeared in the absence of myalgias or muscle weakness but accompanied by marked tiredness and inactivity. She could not eat or drink during four days prior to admission. The patient suspected an adverse effect of trametinib/dabrafenib and discontinued it 2 days prior to admission. Thereafter, she experienced an almost complete remission of the deficits except for ocular muscle weakness and visual impairment.

Los inhibidores de MEX and BRAF como trametinib y dabrafenib se usan en el melanoma metastásico con mutación BRAF, pero pueden tener efectos secundarios. Informamos una paciente de 50 años con un melanoma metastásico con la mutación BRAF que recibió trametinib (2 mg/día) y dabrafenib (200 mg/día) después de usar interferón sin beneficio. Después de iniciar esta terapia la paciente notó una incapacidad de abducir el ojo izquierdo. Ocho días después de iniciar el tratamiento, tuvo falta de apetito, vómitos, diarrea, vértigo y fiebre de 40°C. Dos días después notó pérdida de su agudeza visual, requiriendo asistencia para efectuar sus actividades de vida diaria. Dos días después apareció coluria, en ausencia de mialgias o debilidad muscular, pero acompañadas de fatiga. Ella no pudo comer o tomar líquidos por cuatro días antes de ingresar al hospital. La paciente sospechó que estaba experimentando efectos secundarios de los medicamentos y los suspendió dos días antes del ingreso, experimentando una casi completa remisión de sus síntomas, con excepción de la debilidad de musculatura ocular y déficit visual.

Treatment of chronic hepatitis C in patients with chronic kidney disease with Sofosbuvir-basead regimes

ABSTRACT Background: To analyze the effectiveness and the safety of Sofosbuvir-based regimens to treat patients with chronic hepatitis C virus (HCV) infection and chronic kidney disease (CKD). Methods: A retrospective, observational study in patients with chronic HCV infection and CKD treated with Sofosbuvir-based regimens was performed. Liver fibrosis, comorbidities, HCV genotype and sustained virological resposnse (SVR) at 12th week post-treatment were evaluated. Kidney function was accessed by serum creatinine and glomerular filtration rate (GFR). The assumed level of significance was 5 %. Results: Thirty-five patients were treated. The mean age was 52.1 ± 10.9 years, 19 (54.3 %) were women, 32 (91.4 %) were already kidney transplanted and 3 (8.6 %) were on hemodialysis. The SVR by intention to treat was 88.6 %. The mean GFR was 65.8 ± 28.6 and 63.7 ± 28.3 ml/min pre- and post-treatment respectively (p > 0.05). Treatment was interrupted in 1 (2.85 %) patient due to anemia and in 2 (5.7 %) due to loss of kidney function. Conclusion: Sofosbuvir-based regimens are effective to treat HCV in patients with CKD. In patients with mild CKD this type of therapy seems to be safe.

Tratamento da hepatite C em pacientes com doença renal crônica e pós-tranplante renal: eficácia e segurança dos esquemas de antivirais de ação direta contendo sofosbuvir / Hepatitis c treatment of renal transplant and chronic kidney disease patients: efficacy and safety of direct-acting antiviral regimens containing sofosbuvir

ABSTRACT BACKGROUND: Direct-acting antivirals have revolutionized hepatitis C treatment, also for patients with chronic kidney disease (CKD), but some controversy exists regarding the use of sofosbuvir (SOF) in patients with glomerular filtration rate (GFR) <30 mL/min. OBJECTIVE: To evaluate the efficacy and safety of these regimens for hepatitis C treatment of patients with CKD and after renal transplantation, as well as the impact of SOF on renal function in non-dialysis patients. METHODS: All patients with hepatitis C and CKD or renal transplant treated with direct-acting antivirals at a referral center in Brazil between January 2016 and August 2017 were included. Efficacy was evaluated based on viral load (HCV RNA) and a sustained virological response (SVR) consisting of undetectable RNA 12 and/or 24 weeks after the end of treatment (SVR12 and SVR24) was defined as cure. Safety was determined by adverse events and ribavirin, when combined, was administered in escalating doses to all patients with GFR <60 mL/min. The impact of SOF on renal function was determined by the measurement of baseline creatinine during and after the end of treatment and its increase was evaluated using the Acute Kidney Injury Network (AKIN) classification. RESULTS: A total of 241 patients (52.7% females) with a mean age of 60.72±10.47 years were included. The combination of SOF+daclatasvir was the predominant regimen in 75.6% of cases and anemia was present in 28% of patients who used ribavirin (P=0.04). The SVR12 and SVR24 rates were 99.3% and 97.1%, respectively. The treatment was well tolerated and there were no major clinically relevant adverse events, with the most prevalent being asthenia (57.7%), itching (41.1%), headache (40.7%), and irritability (40.2%). Among conservatively treated and renal transplant patients, oscillations of creatinine levels (AKIN I) were observed in 12.5% of cases during treatment and persisted in only 8.5% after the end of treatment. Of these, 2.0% had an initial GFR <30 mL/min and this percentage decreased to 1.1% after SOF use. Only 0.5% and 1.6% of the patients progressed to AKIN II and AKIN III elevation, respectively. CONCLUSION: The direct-acting antivirals were safe and efficacious in CKD patients treated with SOF-containing regimens, with the observation of high SVR rates, good tolerability and few severe adverse events. The combination with ribavirin increased the risk of anemia and the administration of escalating doses seems to be useful in patients with GFR <60 mL/min. In patients with GFR <30 mL/min, SOF had no significant renal impact, with serum creatinine returning to levels close to baseline after treatment.

RESUMO CONTEXTO: Os antivirais de ação direta revolucionaram o tratamento da hepatite C, inclusive para os pacientes com doença renal crônica (DRC), porém ainda há divergências no emprego do sofosbuvir (SOF) quando taxa de filtração glomerular (TFG) <30 mL/min. OBJETIVO: Avaliar a eficácia e segurança desses esquemas no tratamento da hepatite C em pacientes com DRC e pós-transplante renal, além de avaliar o impacto do SOF sobre a função renal dos não-dialíticos. MÉTODOS: Todos os pacientes com hepatite C e DRC ou transplante renal que realizaram tratamento com antivirais de ação direta em centro referenciado do Brasil no período de janeiro/2016 a agosto/2017 foram incluídos. A eficácia foi avaliada por meio da carga viral (HCV-RNA), considerando-se cura uma resposta virológica sustentada (RVS) com resultado indetectável após 12 e/ou 24 semanas do término do tratamento (RVS12 e RVS24). A segurança foi determinada pelos eventos adversos e a ribavirina, quando associada, foi introduzida de forma escalonada em todos os pacientes com TFG <60 mL/min. Para determinação do impacto do SOF sobre a função renal, foram observadas as dosagens de creatinina basal, durante e após término do tratamento com seu incremento avaliado por meio da classificação de AKIN (acute kidney injury network). RESULTADOS: Foram incluídos 241 pacientes, sendo 52,7% do sexo feminino, com média de idade de 60,72±10,47 anos. A associação de SOF+daclatasvir predominou em 75,6% dos casos e anemia esteve presente em 28% dos pacientes que utilizaram ribavirina (P=0,040). As taxas de RVS12 e RVS24 foram de 99,3% e 97,1%. O tratamento foi bem tolerado, com eventos adversos pouco relevantes, sendo os mais prevalentes: astenia (57,7%), prurido (41,1%), cefaleia (40,7%) e irritabilidade (40,2%). Entre os pacientes em tratamento conservador e transplantados renais, os valores de creatinina sofreram oscilações AKIN I em 12,5% dos casos, durante o tratamento, persistindo em apenas 8,5% da amostra após o término, dos quais 2,0% apresentavam TFG <30 mL/min inicialmente, com queda para 1,1% após uso do SOF. Apenas 0,5% e 1,6% evoluíram com elevação AKIN II e AKIN III. CONCLUSÃO: Os antivirais de ação direta foram seguros e eficazes em pacientes com DRC tratados com esquemas contendo SOF, apresentando altas taxas de RVS, boa tolerabilidade e poucos eventos adversos graves. A associação com ribavirina aumentou o risco de anemia, portanto sua introdução de forma escalonada parece ser útil nos pacientes com TFG <60 mL/min. Em pacientes com TFG <30 mL/min o SOF não apresentou impacto renal significativo, com creatinina sérica retornando a valores próximos ao basal após o tratamento.

Combined ns5a & ns5b nucleotide inhibitor therapy for patients with chronic hepatitis c with stage 5 chronic kidney disease on hemodialysis / Terapia inibidora de nucleotídeo NS5A& NS5B combinada para pacientes com hepatite C crônica com doença renal crônica em estágio 5, em hemodiálise

ABSTRACT BACKGROUND: Hepatitis C virus (HCV) infection is the most common hepatotropic viral infection affecting the patients on maintenance hemodialysis. Treatment of chronic HCV infection in stage 4 and 5 CKD includes a combination of elbasvir/grazoprevir and glecaprevir/pibrentasvir, which are not available in many countries. OBJECTIVE: Hence, we have conducted this study to look for the safety and efficacy of sofosbuvir combination therapy in this difficult to treat population. METHODS: We conducted a single-center, prospective, open-label study in which Stage 5 CKD patients on maintenance hemodialysis with HCV infection. Total of 18 patients was included. sofosbuvir with daclatasvir or ledipasvir was used according to genotype for 12 weeks. HCV RNA, genotype, transient elastography (TE) was considered for every patient. HCV RNA was quantified at 4th week, 12th week and 12 weeks post-treatment to look for sustained virologic response (SVR 12). RESULTS: Infection due to genotype 1 was seen in 12 (66.7%) patients followed by genotype 3 in 4 (22.3%) with each patient of genotype 2 and 5. The median value of HCV RNA was 2,35,000 IU/mL. On TE, all had liver stiffness of <9.4 KPa. All patients had HCV RNA of <15 IU/mL at 4th and 12th week of treatment and 12 weeks post-treatment. No significant change in hemoglobin, eGFR and liver stiffness was observed. CONCLUSION: Full dose sofosbuvir i.e. 400 mg, in combination with NS5A inhibitors daclatasvir or ledipasvir is found to be safe and effective in patients with end stage renal disease, who are on maintenance hemodialysis.

RESUMO CONTEXTO: A infecção pelo vírus da hepatite C (HCV) é a infecção viral hepática mais comum que afeta pacientes em hemodiálise de manutenção. O tratamento da infecção crônica por HCV no estágio 4 e 5 da doença renal crônica inclui uma combinação de elbasvir/grazoprevir e glecaprevir/pibrentasvir, que não estão disponíveis em muitos países. OBJETIVO: Portanto, realizamos este estudo para procurar a segurança e eficácia da terapia combinada de sofosbuvir nesta população de difícil tratamento. MÉTODOS: Realizamos um estudo de centro único, prospectivo e aberto, no qual pacientes com doença renal crônica em estágio 5 em hemodiálise de manutenção com infecção por HCV. Um total de 18 pacientes foi incluído. Sofosbuvir com daclatasvir ou ledipasvir foi usado de acordo com o genótipo por 12 semanas. O HCV RNA, genótipo, elastografia transitória foi considerado para cada paciente. O HCV RNA foi quantificado na 4ª semana, 12ª semana e 12 semanas após o tratamento para procurar uma resposta virológica sustentada. RESULTADOS: A infecção por genótipo 1 foi observada em 12 (66,7%) pacientes, seguido pelo genótipo 3 em 4 (22,3%), em um paciente do genótipo 2 e em outro, 5. O valor mediano do HCV RNA foi de 2.35.000 IU/mL. Na elastografia transitória, todos tinham rigidez hepática de <9.4 KPa. Todos os pacientes tinham RNA HCV <15 IU/mL na 4ª e 12ª semana de tratamento e 12 semanas após o tratamento. Não foi observada nenhuma alteração significativa na hemoglobina, eGFR e rigidez hepática. CONCLUSÃO: A dose completa sofosbuvir ou seja, 400 mg, em combinação com inibidores NS5A daclatasvir ou ledipasvir foi considerada segura e eficaz em pacientes com doença renal em estágio final, que estão em manutenção hemodiálise.

Ponatinib inhibits growth of patient-derived xenograft of cholangiocarcinoma expressing FGFR2-CCDC6 fusion protein in nude mice / 南方医科大学学报

OBJECTIVE@#To investigate the antitumor effect of ponatinib on the growth of cholangiocarcinoma xenograft derived from a clinical patient in a mouse model expressing FGFR2-CCDC6 fusion protein.@*METHODS@#Lung metastatic tumor tissue was collected from a patient with advanced intrahepatic cholangiocarcinoma and implanted subcutaneously a NOD/SCID/ Il2rg-knockout (NSG) mouse. The tumor tissues were harvested and transplanted in nude mice to establish mouse models bearing patient-derived xenograft (PDX) of cholangiocarcinoma expressing FGFR2-CCDC6 fusion protein. The PDX mouse models were divided into 4 groups for treatment with citrate buffer (control group), intragastric administration of 20 mg/kg ponatinib dissolved in citrate buffer (ponatinib group), weekly intraperitoneal injections of 50 mg/kg gemcitabine and 2.5 mg/ kg cisplatin (gemcitabine group), or ponatinib combined with gemcitabine and cisplatin at the same doses (10 mice in each group, and 9 mice were evaluated in ponatinib group). The expressions of p-FGFR, p-FRS2, p-AKT, p-ERK, CD31, and Ki-67 in the xenografts were evaluated with immunohistochemistry, and cell apoptosis was analyzed with cleaved caspase-3 (CC3) staining and TUNEL staining. Western blotting was used to detect the expressions of FGFR2, p-FGFR, AKT, p-AKT, ERK, p-ERK, FRS2 and p-FRS2 in the tumor tissues.@*RESULTS@#Compared with those in the control group, the mice in ponatinib group showed a significantly reduced tumor volume (@*CONCLUSIONS@#Ponatinib can regulate FGFR signaling to inhibit the proliferation and induce apoptosis of tumor cells in mice bearing patient-derived cholangiocarcinoma xenograft with FGFR2 fusion. FGFR inhibitor can serve as a treatment option for patients with cholangiocarcinoma with FGFR2 fusion.

Olmesartan inhibits age-associated migration and invasion of human aortic vascular smooth muscle cells by upregulating miR-3133 axis / 南方医科大学学报

OBJECTIVE@#To explore the effects of olmesartan on age-associated migration and invasion capacities and microRNA (miRAN) axis in human aortic vascular smooth muscle cells (HA-VSMCs).@*METHODS@#Cultured HA-VSMCs were divided into control group, bleomycin-mediated senescence (BLM) group and bleomycin + olmesartan treatment group. Wound-healing assay and Boyden chambers invasion assay were used to assess the changes in migration and invasion of the cells, gelatin zymography was used to analyze matrix metalloproteinase-2 (MMP-2) activation in the cells. The differentially expressed miRNAs were identified by miRNA microarray assay and validated by quantitative real-time PCR. MiR-3133 inhibitor was used to examine the effects of molecular manipulation of olmesartan on age-associated migration and invasion and MMP-2 activation in the cells.@*RESULTS@#Compared with those of the control group, the percentage of the repopulated cells and the number of cells crossing the basement membrane increased significantly in BLM group [(78.43±12.76)% (42.47±7.22)%, < 0.05; 33.33±5.51 13.00±4.36, < 0.05]. A significant increase of MMP-2 activation was found in BLM group as compared with the control group (1.66 ± 0.27 0.87 ± 0.13, < 0.05). Olmesartan significantly inhibited BLM-induced enhancement of cell migration and invasion and MMP-2 secretion in the cells. MiR-3133 was significantly downregulated in BLM group and upregulated in olmesartan group. Transfection with miR-3133 inhibitor significantly reversed the effects of olmesartan on age-associated migration and invasion of the cells [(85.87±7.39)% (49.77±3.05)%; 34.67±2.31 20.00±4.58, < 0.05] and MMP-2 activation in the cells (1.76±0.19 0.94±0.10, < 0.05).@*CONCLUSIONS@#Olmesartan inhibits the migration and invasion of ageassociated HA-VSMCs probably by upregulating of the miR-3133 axis.

Effect of 5-aminoimidazole-4-formamide Ribonucleotide Combined with Interferon on Chronic Myeloid Leukemia K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To study the effect of 5-aminoimidazole-4-formamide ribonucleotide (AICAR) combined with interferon (IFN-α-2b) on the proliferation and apoptosis of chronic myeloid leukemia K562 cells, and explore its possible mechanism.@*METHODS@#CCK-8 method was used to detect the inhibition of cell proliferation. Wright Giemsa method was used to stain and cell morphology was observed by light microscopy. FITC Annexin V/PI double staining method was used to analyze the change of apoptosis rate. Immunocytochemistry method was used to detect the expression of wild-type P53 protein.@*RESULTS@#Different concentration of AICAR was inhibitory effect on K562 cells at different time point of action for 24 h, 48 h, and 72 h, and the inhibition was time and dose-dependent (r=0.71, r=0.84). The combination of AICAR and IFN-α-2b could effectively inhibit the proliferation and promote apoptosis of K562 cells. The inhibition rate of K562 cells was (45.26±2.54)%, and the early apoptosis rate was (33.72±0.23)%, which was statistically significantly different from the control group, AICAR or IFN-ɑ-2b alone (P<0.05). The combination of two drugs promoted the expression of wild-type p53 protein.@*CONCLUSION@#AICAR and/or IFN-ɑ-2b can inhibit the cell proliferation and promote the apoptosis of K562 cells. The combination of two drugs shows synergistic antitumor effect, and its mechanism may be related to the promotion of high expression of wild-type p53 protein.

Protective effect of necrostatin1 on the damage of pancreas islet cells induced by TNFα / 中南大学学报(医学版)

OBJECTIVES@#To investigate whether necrostatin-1 (Nec-1) can protect islet cells from the damage induced by TNF-α.@*METHODS@#After isolation and purification, the neonatal porcine islet cell clusters (NICCs) were divided into 3 groups (islets 10 000 IEQ/group): a Nec-1 group (Nec-1+TNF-α was added to the culture medium), a TNF-α group (TNF-α was added to the culture medium), and a control group (pure medium). The number of cells was observed after 48 h of co-culture. The cell death was evaluated by AO/EB staining. Insulin secretion and DNA of islets were detected by chemiluminescence and nucleic acid quantitative analysis. RT-PCR assay was used to examine the mRNA expressions of insulin gene, glueogan gene and somatostatin gene. Flow cytometry analysis was used to detect the viability of B cells.@*RESULTS@#The number of islets in Nec-1 group, TNF-α group and the control group were (8 425±2 187), (4 325±778), and (7 122±1 558) IEQ, respectively. Compared to the other two groups, the number of dead cells in TNF-α group was greatly increased. The insulin/DNA values in the Nec-1 group, TNF-α group and blank control group were (13.21±3.15), (2.47±0.45), and (7.44±0.97) mIU/mg, respectively. Compared to the TNF-α group and the control group, the mRNA relative expression levels of insulin gene (6.73±1.07), glucagon gene (10.13±1.98), somatostatin gene (8.57±1.11) were significantly increased in the Nec-1 group (all <0.05), the rate of live cells (97.32±1.87)% and live B cells (90.86±3.68)% were increased significantly in the Nec-1 group (all <0.05).@*CONCLUSIONS@#TNF-α can induce neonatal porcine islet cells damage, which is attenuated in the presence of Nec-1. Nec-1 can increase the content of endocrine cells in NICCs.

Paniculitis secundaria a terapia target en paciente con melanoma, lo que el dermatólogo debe saber: reporte de un caso / Panniculitis in association with target therapy in melanoma patient, what the dermatologist should know: a case report

Las terapias target constituyen hoy en día una alternativa terapéutica cada vez más utilizada para el manejo de pacientes con melanoma metastásico. Sin embargo, se han descrito múltiples efectos farmacológicos adversos asociados a su uso, siendo los cutáneos los de mayor prevalencia. Se presenta el caso de un hombre de 55 años con diagnóstico de melanoma cutáneo metastásico etapa IV, BRAFV600E mutado, en tratamiento con dabrafenib/trametinib que consultó por desarrollo de lesiones nodulares eritematosas sensibles en extremidades superiores e inferiores, asociadas a sensación febril durante el curso del tratamiento. Se descartó alguna infección sobreagregada. Se realizó una biopsia de las lesiones cutáneas, con confirmación diagnóstica histopatológica de una paniculitis mixta de predominio septal, granulomatosa y con vasculitis leucocitoclástica. La paniculitis asociada a esta terapia ha sido descrita en la literatura y se ha considerado un efecto farmacológico inmunomediado adverso, relacionándose a un mejor pronóstico para el melanoma metastásico en tratamiento. Por lo tanto, así como en el caso presentado, se evita la suspensión del fármaco y se asocia terapia sintomática en caso de mayores molestias del paciente. Es de alta relevancia para el dermatólogo conocer e interpretar adecuadamente este efecto adverso farmacológico, y así indicar el manejo más adecuado para el paciente.

Target therapies are currently a therapeutic option increasingly used for the management of patients with metastatic melanoma. However, there are multiple adverse pharmacological effects associated with their use that have been described. Cutaneous adverse reactions are the most frequent. We report the case of a 55-year-old man with a diagnosis of stage IV BRAFV600E-mutated metastatic cutaneous melanoma undergoing treatment with dabrafenib/trametinib, who consulted due to the development of erythematous nodular lesions in the upper and lower limbs associated with febrile sensation during the course of treatment. Infection was ruled out and a biopsy of the skin lesions was done, which provided the histopathological confirmation of a predominantly septal, granulomatous with leukocytoclastic vasculitis, mixed panniculitis. Panniculitis associated with this therapy has been described in the literature and has been considered an immune-mediated pharmacological adverse effect. It is considered to be related to a better prognosis in the treatment of metastatic melanoma. Consequently, as shown in this case report, target therapy should not be discontinued and symptomatic medication should be given to alleviate patient discomfort. The dermatologist should know and properly interpret this adverse effect and prescribe the most appropriate management for the patient.

P38 MAPK inhibitor SB203580 attenuates the toxicity of ropivacaine on PC12 cells / 中南大学学报(医学版)

To investigate the effect of SB203580, a p38MAPK specific inhibitor, on ropivacaine-induced cytotoxicity in PC12 cells.
 Methods: PC12 cells were divided into three groups: the normal group (Group N), cells were cultured for 48 h; the ropivacaine group (Group R), cells were cultured with 15 mmol/L ropivacaine hydrochloride for 48 h; the ropivacaine+SB203580 group (Group R+S), cells were cultured with 15 mmol/L ropivacaine hydrochloride plus 10 μmol/L SB203580 for 48 h. The cell survival rates were detected by MTT assay. The protein levels of cleaved caspase-3, phosphor-p38 (p-p38) and cystolic cytochrome C (Cyt C) were detected by Western blotting.
 Results: Compared with the Group N, the number and survival rate of PC12 cells in the Group R and the Group R+S were significantly reduced (all P<0.05); the number and survival rate of PC12 cells in the Group R+S were significantly higher than those in the Group R (both P<0.05). Compared with the Group N, the levels of p-p38 and cleaved caspase-3, and the content of cytoplasmic Cyt C in the PC12 cells from the Group R and the Group R+S were significantly enhanced (all P<0.05); compared with the Group R, the levels of p-p38 and cleaved caspase-3, and the content of cytoplasmic Cyt C in the PC12 cells from the Group R+S were decreased (all P<0.05).
 Conclusion: The ropivacaine-induced cytotoxicity can be attenuated via inhibition of p38MAPK; which is related to decrease in Cyt C content and cleaved caspase-3 expression.

Kir2.1 Channel Regulation of Glycinergic Transmission Selectively Contributes to Dynamic Mechanical Allodynia in a Mouse Model of Spared Nerve Injury / 神经科学通报·英文版

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.

Clinical characteristics of chronic myeloid leukemia with T315I mutation and the efficacy of ponatinib / 南方医科大学学报

OBJECTIVE@#To analyze the clinical features of chronic myeloid leukemia (CML) with T315 I mutation (CML-T315I) and compare the effectiveness of different treatments.@*METHODS@#We retrospectively analyzed the clinical data and outcomes of 19 patients with CML-T315I receiving different treatments. The T315 I mutations in these patients were detected by examination of BCR-ABL kinase domain (KD) mutation by RTQ-PCR and Sanger sequencing. The relapse following the treatments, defined as hematological, cytogenetic and molecular biological recurrences, were analyzed in these patients.@*RESULTS@#Of the 19 patients with CML-T315I, 14 (73.7%) were in CML-CP stage at the initial diagnosis, and 13 (81.2%) were high-risk patients based on the Sokal scores. All the 19 patients were treated with TKI after the initial diagnosis, and during the treatment, 15 (78.9%) patients were found to have additional chromosomal aberrations, and 10 (52.6%) had multiple mutations; 13 (68.4%) of the patients experienced disease progression (accelerated phase/blast crisis) before the detection of T315I mutation, with a median time of 40 months (5-120 months) from the initial diagnosis to the mutation detection. After detection of the mutation, 12 patients were treated with ponatinib and 7 were managed with the conventional chemotherapy regimen, and their overall survival rates at 3 years were 83.3% and 14.2%, respectively ( < 0.001).@*CONCLUSIONS@#CML patients resistant to TKI are more likely to have T315I mutations, whose detection rate is significantly higher in the progressive phase than in the chronic phase. These patients often have additional chromosomal aberrations and multiple gene mutations with poor prognoses and a high recurrence rate even after hematopoietic stem cell transplantation. Long-term maintenance therapy with ponatinib may improve the prognosis and prolong the survival time of the patients.

Prostaglandin E receptors differentially regulate the output of proinflammatory cytokines in myometrial cells from term pregnant women / 生理学报

Prostaglandin (PG) E plays critical roles during pregnancy and parturition. Emerging evidence indicates that human labour is an inflammatory event. We sought to investigate the effect of PGE on the output of proinflammatory cytokines in cultured human uterine smooth muscle cells (HUSMCs) from term pregnant women and elucidate the role of subtypes of PGE receptors (EP, EP, EP and EP). After drug treatment and/or transfection of each receptor siRNA, the concentrations of inflammatory secreting factors in HUSMCs culture medium were detected by the corresponding ELISA kits. The results showed that, PGE increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) output, decreased chemokine (c-x-c motif) ligand 8 (CXCL8) output in a dose-dependent manner, but had no effect on IL-1β and chemokine (c-c motif) ligand 2 (CCL-2) secretion of HUSMCs. EP/EP agonist 17-phenyl-trinor-PGE stimulated IL-6 and TNFα whilst suppressing IL-1β and CXCL8 output. The effects of 17-phenyl-trinor-PGE on IL-1β and CXCL8 secretion were remained whereas its effect on IL-6 and TNFα output did not occur in the cells with EP knockdown. The stimulatory effects of 17-phenyl-trinor-PGE on IL-6 and TNFα were remained whereas the inhibitory effects of 17-phenyl-trinor-PGE on IL-1β secretion was blocked in the cells with EP knockdown. Either of EP and EP agonists stimulated IL-1β and TNFα output, which was reversed by EP and EP siRNA, respectively. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked EP/EP modulation of TNFα and CXCL8 output. PI3K inhibitor LY294002 and P38 inhibitor SB202190 blocked 17-phenyl-trinor-PGE-induced IL-1β and IL-6 output, respectively. The inhibitors of adenylyl cyclase and PKA prevented EP and EP stimulation of IL-1β and TNFα output, whereas PLC and PKC inhibitors blocked EP- and EP-induced TNFα output but not IL-1β output. Our data suggest that PGE receptors exhibit different effects on the output of various cytokines in myometrium, which can subtly modulate the inflammatory microenvironment in myometrium during pregnancy.

Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.