Background and Aims:Carotid plaque instability is a critical pathological basis for ischemic stroke.Identifying key molecular markers to evaluate plaque stability has important clinical implications.Recent studies have emphasized the regulatory roles and predictive value of long non-coding RNAs(lncRNAs)in plaque stability.In our previous transcriptome sequencing analysis of human stable and unstable carotid plaques,we identified lncRNA C-C motif chemokine ligand 3 antisense RNA 1(CCL3-AS1)as significantly upregulated in unstable plaques,suggesting a potential association with plaque instability.Therefore,this study aimed to validate CCL3-AS1 expression in an expanded plaque sample cohort and to explore its role and underlying molecular mechanism in carotid plaque destabilization.Methods:Carotid plaque specimens were obtained from patients undergoing carotid endarterectomy and classified into stable and unstable groups(n=15 per group)based on HE and Sirius red staining.qRT-PCR was used to validate the expression of candidate lncRNA CCL3-AS1.The localization and co-expression of CCL3-AS1 with macrophages in plaques were determined by RNA fluorescence in situ hybridization(FISH)combined with immunofluorescence staining.In vitro,THP1-derived macrophages were transduced with lentivirus or treated with antisense oligonucleotides(ASO)to overexpress or knock down CCL3-AS1,respectively,and the expression levels of inflammatory cytokines and matrix metalloproteinases(MMPs)were assessed.In vivo,an unstable carotid plaque model was established by tandem ligation of the right carotid artery in apolipoprotein E-deficient(ApoE-/-)mice,followed by local overexpression of CCL3-AS1.The effects on plaque morphology,macrophage infiltration,and MMP-9 expression were evaluated.Additionally,bioinformatic prediction using the catRAPID v2.1 omics platform was performed to identify potential RNA-binding proteins interacting with CCL3-AS1.RNA stability assays and RNA-binding protein immunoprecipitation(RIP)were conducted to verify the regulatory mechanism of MMP-9 expression.Results:CCL3-AS1 was significantly upregulated in unstable carotid plaques and was predominantly localized to the cytoplasm of plaque-infiltrating macrophages.In vitro,overexpression of CCL3-AS1 markedly increased the expression of MCP-1,TNF-α,IL-1β,iNOS,and MMP-9 in macrophages,whereas knockdown had the opposite effect.In the ApoE-/-mouse model of unstable carotid plaques,CCL3-AS1 overexpression led to fibrous cap rupture,increased infiltration of pro-inflammatory macrophages,enhanced MMP-9 secretion,and promoted plaque instability.Co-expression analysis revealed a strong correlation between CCL3-AS1 and MMP-9 expression(r=0.89,P=0.001).RNA stability assays demonstrated that CCL3-AS1 delayed the degradation of MMP-9 mRNA.Bioinformatic prediction identified heterogeneous nuclear ribonucleoprotein K(hnRNP-K)as a potential binding partner of CCL3-AS1.RIP and FISH co-localization confirmed the interaction,suggesting that CCL3-AS1 enhances MMP-9 mRNA stability through binding to hnRNP-K,thereby promoting its expression.Conclusion:As a macrophage-enriched inflammatory lncRNA,CCL3-AS1 may promote carotid plaque instability by enhancing MMP-9 expression via hnRNP-K-mediated mRNA stabilization.This lncRNA represents a potential molecular target for early intervention and stratification of ischemic stroke.

Objective To investigate the risk factors for acute respiratory failure in immunocompromised patients with Pneumocystis jirovecii pneumonia(PJP).Methods Clinical data of 123 immunocompromised patients complicated with PJP hospitalized at Mengchao Hepatobiliary Hospital of Fujian Medical University from January 2021 to December 2023 were retrospectively collected and analyzed.SPSS 22.0 statistical software package was used to perform multivariate binary logistic regression analysis to identify risk factors for acute respiratory failure in PJP patients.Results Among the 123 PJP patients,77 were HIV-positive,and 46 were HIV-negative.HIV-negative PJP patients were more likely to have comorbidities such as hypertension(P＜0.001),diabetes mellitus(P＜0.001),coronary heart disease(P=0.034),chronic kidney disease(P＜0.001),chronic liver disease(P=0.019),chronic lung disease(P=0.011),and malignant tumor(P＜0.001).They were also more prone to respiratory failure(P＜0.001)and ICU admission(P＜0.001).The HIV-positive patients had significantly lower CD4+T lymphocyte counts and albumin levels(P＜0.001).Forty patients developed acute respiratory failure,and six patients died.Multivariate analysis showed that high neutrophil-to-lymphocyte ratio(NLR)(P=0.031),non-HIV infection(P=0.002),and concomitant infections with other pathogens(P＜0.001)were independent risk factors for incidence of respiratory failure.ROC curve analysis revealed that the area under the curve(AUC)was 0.686(0.584,0.789)for non-HIV infection,0.731(0.637,0.826)for concomitant infections with other pathogens,0.648(0.546,0.750)for NLR.The predicted probability was 0.845(0.778,0.912).Conclusions Non-HIV infection,high NLR,and concomitant infections with other pathogens are independent risk factors for incidence of respiratory failure in PJP patients.The panel combining these factors provides a higher predictive value for respiratory failure.Timely assessment of patient condition and early treatment are vital for better outcomes.

Background and Aims:Carotid plaque instability is a critical pathological basis for ischemic stroke.Identifying key molecular markers to evaluate plaque stability has important clinical implications.Recent studies have emphasized the regulatory roles and predictive value of long non-coding RNAs(lncRNAs)in plaque stability.In our previous transcriptome sequencing analysis of human stable and unstable carotid plaques,we identified lncRNA C-C motif chemokine ligand 3 antisense RNA 1(CCL3-AS1)as significantly upregulated in unstable plaques,suggesting a potential association with plaque instability.Therefore,this study aimed to validate CCL3-AS1 expression in an expanded plaque sample cohort and to explore its role and underlying molecular mechanism in carotid plaque destabilization.Methods:Carotid plaque specimens were obtained from patients undergoing carotid endarterectomy and classified into stable and unstable groups(n=15 per group)based on HE and Sirius red staining.qRT-PCR was used to validate the expression of candidate lncRNA CCL3-AS1.The localization and co-expression of CCL3-AS1 with macrophages in plaques were determined by RNA fluorescence in situ hybridization(FISH)combined with immunofluorescence staining.In vitro,THP1-derived macrophages were transduced with lentivirus or treated with antisense oligonucleotides(ASO)to overexpress or knock down CCL3-AS1,respectively,and the expression levels of inflammatory cytokines and matrix metalloproteinases(MMPs)were assessed.In vivo,an unstable carotid plaque model was established by tandem ligation of the right carotid artery in apolipoprotein E-deficient(ApoE-/-)mice,followed by local overexpression of CCL3-AS1.The effects on plaque morphology,macrophage infiltration,and MMP-9 expression were evaluated.Additionally,bioinformatic prediction using the catRAPID v2.1 omics platform was performed to identify potential RNA-binding proteins interacting with CCL3-AS1.RNA stability assays and RNA-binding protein immunoprecipitation(RIP)were conducted to verify the regulatory mechanism of MMP-9 expression.Results:CCL3-AS1 was significantly upregulated in unstable carotid plaques and was predominantly localized to the cytoplasm of plaque-infiltrating macrophages.In vitro,overexpression of CCL3-AS1 markedly increased the expression of MCP-1,TNF-α,IL-1β,iNOS,and MMP-9 in macrophages,whereas knockdown had the opposite effect.In the ApoE-/-mouse model of unstable carotid plaques,CCL3-AS1 overexpression led to fibrous cap rupture,increased infiltration of pro-inflammatory macrophages,enhanced MMP-9 secretion,and promoted plaque instability.Co-expression analysis revealed a strong correlation between CCL3-AS1 and MMP-9 expression(r=0.89,P=0.001).RNA stability assays demonstrated that CCL3-AS1 delayed the degradation of MMP-9 mRNA.Bioinformatic prediction identified heterogeneous nuclear ribonucleoprotein K(hnRNP-K)as a potential binding partner of CCL3-AS1.RIP and FISH co-localization confirmed the interaction,suggesting that CCL3-AS1 enhances MMP-9 mRNA stability through binding to hnRNP-K,thereby promoting its expression.Conclusion:As a macrophage-enriched inflammatory lncRNA,CCL3-AS1 may promote carotid plaque instability by enhancing MMP-9 expression via hnRNP-K-mediated mRNA stabilization.This lncRNA represents a potential molecular target for early intervention and stratification of ischemic stroke.

Objective To investigate the risk factors for acute respiratory failure in immunocompromised patients with Pneumocystis jirovecii pneumonia(PJP).Methods Clinical data of 123 immunocompromised patients complicated with PJP hospitalized at Mengchao Hepatobiliary Hospital of Fujian Medical University from January 2021 to December 2023 were retrospectively collected and analyzed.SPSS 22.0 statistical software package was used to perform multivariate binary logistic regression analysis to identify risk factors for acute respiratory failure in PJP patients.Results Among the 123 PJP patients,77 were HIV-positive,and 46 were HIV-negative.HIV-negative PJP patients were more likely to have comorbidities such as hypertension(P＜0.001),diabetes mellitus(P＜0.001),coronary heart disease(P=0.034),chronic kidney disease(P＜0.001),chronic liver disease(P=0.019),chronic lung disease(P=0.011),and malignant tumor(P＜0.001).They were also more prone to respiratory failure(P＜0.001)and ICU admission(P＜0.001).The HIV-positive patients had significantly lower CD4+T lymphocyte counts and albumin levels(P＜0.001).Forty patients developed acute respiratory failure,and six patients died.Multivariate analysis showed that high neutrophil-to-lymphocyte ratio(NLR)(P=0.031),non-HIV infection(P=0.002),and concomitant infections with other pathogens(P＜0.001)were independent risk factors for incidence of respiratory failure.ROC curve analysis revealed that the area under the curve(AUC)was 0.686(0.584,0.789)for non-HIV infection,0.731(0.637,0.826)for concomitant infections with other pathogens,0.648(0.546,0.750)for NLR.The predicted probability was 0.845(0.778,0.912).Conclusions Non-HIV infection,high NLR,and concomitant infections with other pathogens are independent risk factors for incidence of respiratory failure in PJP patients.The panel combining these factors provides a higher predictive value for respiratory failure.Timely assessment of patient condition and early treatment are vital for better outcomes.

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord

Objective To investigate the effects of disulfiram (DS) combined with Cu on the human Burkitt lymphoma cell xenografts in nude mice.Methods Burkitt lymphoma xenograft was established by subcutaneous injection of Raji cell into nude mice after 2 Gy whole body X-irradiation (1×107 Raji cells were resuspended in 200 μl saline).18 bearing tumor mice were randomly divided into control group,DS group and DS/Cu group.During the experiment,the effects of DS/Cu on the nude mice with tumors were examined,including the tumor volumes,weights and the growth curves of xenograft tumor.Histopathological examination of tumor tissue was observed with optical microscape.The protein expression levels of p-JNK and c-jun were also detected by Western blot.Results Subsequent tumor size and weight in DS or DS/Cu-treated animals were (67.71±2.15) mm3,(33.35±7.74) mm3 and (43.35±4.22) mg,(18.05±2.88) mg.One-way ANOVA analysis indicated that the tumor size and weight in DS or DS/Cu-treated animals were reduced significantly relative to tumors in vehicle-treated animals (F =27.579,P =0.000;F =16.369,P =0.000).Furthermore,multiple comparisons revealed that the DS or DS/Cu-treated animals had significantly reduced tumor size and weight compared with control animals (all P ＜ 0.05).There were significant differences in tumor size and weight between DS or DS/Cu-treated animals (both P ＜ 0.05).Tumor inhibition rates in DS or DS/Cu group were 63.48 ％ and 80.24 ％,respectively.An increase of apoptosis changes in the xenograft tumor cells in DS or DS/Cu treated mice were more significant.Westem blot showed that the p-JNK and c-jun protein expressions in the tumors were improved after the DS or DS/Cu treatment,more obvious in DS/Cu treatment.Conclusion DS/Cu can inhibit the growth of xenografts,and one possible mechanism may involve the regulation of JNK signal pathway.

OBJECTIVETo observe the clinical characteristics of infections in adult acute leukemia （AL）patients during chemotherapy in hospital, and identify the risk factors for infections.

METHODSA retrospective study of patients with AL who underwent chemotherapy between July 2010 and Dec 2014 in the First Affiliated Hospital of Xiamen University was conducted. Clinical features and risk factors for infections were analyzed.

RESULTS191 patients with AL received a total of 728 courses of chemotherapies. During these admissions, 385（52.9%) infections episodes occurred. The common infections sites were lower respiratory tract infection（36.3%，153/374）, bloodstream infection（17.1%, 64/374）, oral infection（13.6%，51/374）, and perianal infection（13.4%, 50/374）. 164 strains of pathogenic bacteria were detected. Gram- negative bacteria were recorded in 59.1% of documented pathogens, and Gram- positive bacteria were responsible for 32.9% of infections. Multivariate unconditioned logistic analysis of factors identified consistent independent risk factors for no completely remission（OR=0.142, P< 0.001）, duration of neutropenia longer than 7 days（OR=12.764, P<0.001）, general wards（OR=1.821, P< 0.001）, and hospitalization interval longer than 10 days（OR=0.720, P=0.039）.

CONCLUSIONInfections after chemotherapy for AL continues to be common. AL patients with induction chemotherapy or severe neutropenia faced an increased risk of infections by multivariate analysis. And patients with short-term stay or laminar flow wards seem to be less susceptible to infections.

Acute Disease ; Bacterial Infections ; complications ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hospitals ; Humans ; Leukemia ; complications ; drug therapy ; microbiology ; Multivariate Analysis ; Neutropenia ; complications ; Remission Induction ; Retrospective Studies ; Risk Factors

OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

Cell Culture Techniques ; Cell Differentiation ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocyte Progenitor Cells ; cytology