[Predictive value of lipoproteins on progression to chronic critical illness in intensive care unit patients].

OBJECTIVE: To explore the predictive value of lipoproteins on the progression of critically ill patients to chronic critical illness (CCI). METHODS: A retrospective cohort study was conducted to analyze clinical data of patients admitted to the intensive care unit (ICU) of Nanjing Drum Tower Hospital from January 1, 2020, to December 31, 2022. The levels of high-density lipoprotein (HDL), low-density lipoprotein (LDL) and apolipoproteins (ApoA-I, ApoB) at 1, 3, 7, 14 and 21 days after admission to ICU were collected. The progression to CCI was recorded. CCI was defined as the length of ICU stay ≥14 days with sustained organ dysfunction [sequential organ failure assessment (SOFA) score ≥2]. Differences in lipoprotein levels between the patients with and without CCI were compared. Multivariate Logistic regression was used to analyze risk factors for critically ill patients progressing to CCI. Receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of lipoproteins on critically ill patients progressing to CCI. RESULTS: A total of 200 patients were enrolled in the final analysis. 137 patients (68.5%) progressed to CCI, and 63 patients (31.5%) did not. The lipoprotein indicators in the CCI group showed a decrease after the acute phase, while the lipoprotein indicators in the non-CCI group showed an increase. The levels of HDL, LDL, ApoA-I, and ApoB at various time points in the CCI group were significantly lower than those in the non-CCI group. HDL at 7 days in the CCI group was significantly lower than that in the non-CCI group [mmol/L: 0.44 (0.31, 0.61) vs. 0.67 (0.49, 0.75), P < 0.01]. Multivariate Logistic regression analysis showed that 7-day HDL was an independent risk factor for critically ill patients progressing to CCI [odds ratio (OR) = 0.033, 95% confidence interval (95%CI) was 0.004-0.282, P = 0.002]. ROC curve analysis showed that the area under the ROC curve (AUC) of 7-day HDL for predicting critically ill patients progressing to CCI was 0.702, with a 95%CI of 0.625-0.779, P < 0.001. When the optimal cut-off value was 0.59 mmol/L, the sensitivity was 69.8%, and the specificity was 72.4%. CONCLUSIONS: The low level of lipoproteins is closely related to the progression of critically ill patients, and 7-day HDL has a certain predictive value for critically ill patients progressing to CCI. Continuously observation of the change trend of lipoprotein level is helpful to judge the progression of CCI in critically ill patients.

Corrigendum: Acute gastrointestinal injury and altered gut microbiota are related to sepsis-induced cholestasis in patients with intra-abdominal infection: a retrospective and prospective observational study.

[This corrects the article DOI: 10.3389/fmed.2023.1144786.].

Acute gastrointestinal injury and altered gut microbiota are related to sepsis-induced cholestasis in patients with intra-abdominal infection: a retrospective and prospective observational study.

Background: Sepsis-associated liver dysfunction (SALD) has high incidence and mortality in patients with intra-abdominal infection (IAI). The associations between acute gastrointestinal injury (AGI), gut microbiota, and SALD were evaluated in patients with IAI. Methods: A retrospective study was conducted to assess the relationship between AGI and SALD in patients with IAI. Patients were divided into non-SALD and sepsis-induced cholestasis (SIC) groups, which is a subtype of SALD. SIC was defined as total bilirubin >2 mg/dL. AGI incidences between the two groups were compared using Chi-square test. Subsequently, a prospective study was conducted to investigate the gut microbiota differences between patients without SALD and those with SIC. Fecal samples were collected on days 1, 3, and 7 after admission to analyze changes in gut microbiota using 16S ribosomal ribonucleic acid sequencing. Results: One hundred thirty-four patients with IAI were included retrospectively, with 77 SALD and 57 non-SALD cases. Among patients with SALD, 71 were diagnosed with SIC. Patients with SIC had a higher incidence of AGI compared to those without SALD (28.07% vs. 56.34%, p < 0.05), and a severity-dependent relationship was found between AGI grade and SIC occurrence. Subsequently, 20 patients with IAI were recruited prospectively, with 10 patients each assigned to the non-SALD and SIC groups. Patients with SIC had a more severe gut microbiota disorder on day 7 than those without SALD, including lower microbiota diversities, decreased abundance of Firmicutes and Bacteroidetes, and increased abundance of Proteobacteria and Actinobacteria at the phylum level. Furthermore, Burkholderia - Caballeronia - Paraburkholderia and Delftia, the two most abundant genera, were significantly higher in the SIC group than in the non-SALD group. Functional prediction analysis showed that the top three KEGG pathways were ribosome, pyrimidine metabolism, and the two-component system. During the first week, the abundance of Proteobacteria decreased significantly, whereas Cyanobacteria increased in the non-SALD group; however, the phyla taxa did not change significantly in the SIC group. Conclusion: There exists a severity-dependent relationship between AGI grade and SIC occurrence in adult patients with IAI. A severe gut microbiota disorder was discovered in SIC during the first week of the intensive care unit stay.

A novel mini-invasive step-up approach for the treatment of severe acute pancreatitis with extensive infected necrosis: A single center case series study.

RATIONALE: Complete removal of necrosis is critical for treating patients with severe acute pancreatitis (SAP) presenting infection of pancreatic necrosis (IPN). Frequently used mini-invasive methods include the surgical step-up approach suitable for necrosis extending laterally, whereas the endoscopic step-up approach is suitable for medial necrosis. However, in patients with extensive IPN, either approach alone usually has limited treatment effects. PATIENT CONCERNS: We describe a case series of combined mini-invasive step-up approach for treating extensive IPN. DIAGNOSES: Patients were diagnosed with SAP and had extensive IPN. INTERVENTIONS: Seven patients with SAP and extensive IPN were enrolled. All patients underwent a combined step-up approach comprising 4 steps: percutaneous catheter drainage, continuous negative pressure irrigation (CNPI), percutaneous endoscopic necrosectomy (PEN), and transgastric necrosectomy (TN). OUTCOMES: The median interval from symptom onset to percutaneous catheter drainage and CNPI was 11 days (range, 6-14) and 18 days (range, 14-26), and the median CNPI duration was 84 days (range, 54-116). The median interval from the onset of symptoms to PEN and TN was 36 days (range, 23-42) and 41 days (range, 34-48), respectively, and the median number of procedures was 2 (range, 1-2) for PEN and 3 (range, 2-4) for TN. Only a minor case of abdominal bleeding and a pancreatic-cutaneous fistula were reported, both resolved after conservative treatment. The median length of stay in the intensive care unit was 111 days (range, 73-133); all patients survived. LESSONS: This mini-invasive step-up approach shows promising clinical effects and is relatively safe in critically ill patients with extensive IPN and high-risk surgical intervention.

LIPOPOLYSACCHARIDE-PRECONDITIONED MESENCHYMAL STEM CELL TRANSPLANTATION ATTENUATES CRITICAL PERSISTENT INFLAMMATION IMMUNE SUPPRESSION AND CATABOLISM SYNDROME IN MICE.

ABSTRACT: Background: Persistent inflammation, immunosuppression, and catabolism syndrome (PIICS) is associated with high mortality and high health care costs, and there is currently no effective target treatment. Mesenchymal stem cells (MSCs) possess multipotent immunomodulatory properties. LPS-preconditioned type 1 MSCs (MSC1s) are potentially beneficial for PIICS treatment because of their proinflammatory, anti-infective, and healing properties. Here, we investigated the therapeutic efficacy and mechanisms of action of MSC1s in PIICS. Methods: We previously optimized a reaggravated PIICS mouse model, which was used in this study. PIICS mice were subjected to cecal ligation and puncture on day 1 and LPS injection on day 11. Subsequently, the mice were treated with or without MSC1s. Animal survival and phenotypes, along with the levels of catabolism, inflammation, and immunosuppression, were evaluated. MSC1s were cocultured with CD8 + T cells in vitro , and inflammatory cytokine levels and CD8 + T-cell function were assessed. Results: MSC1 transplantation alleviated weight loss and muscle wasting, inhibited catabolism and inflammation, and considerably improved the proportion and function of CD8 + T cells in the PIICS mice. After coculture with MSC1s, the expression levels of CD107a and interferon γ increased, whereas the expression level of programmed death 1 decreased significantly in CD8 + T cells. MSC1s also promoted proinflammatory cytokine secretion and reduced the concentration of soluble PD-L1 in vitro . Conclusions: MSC1s can protect mice against critical PIICS, partly by enhancing CD8 + T-cell function. Therefore, MSC1 transplantation is a novel therapeutic candidate for PIICS.

Association of Monocyte-to-Lymphocyte and Neutrophil-to-Lymphocyte Ratios With Persistent Critical Illness in Patients With Severe Trauma.

BACKGROUND: Neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio are reported to reflect the inflammation and immune status in critically ill patients, but their role in severe trauma patients with persistent critical illness remains to be elucidated. OBJECTIVE: We aimed to evaluate the relationship of neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio with persistent critical illness in severe trauma patients. METHODS: In a single-center retrospective cohort study, persistent critical illness was defined as intensive care unit length of stay of more than 10 days. Monocyte-to-lymphocyte ratio and neutrophil-to-lymphocyte ratio were computed individually and categorized into 3 tertiles. Logistic regression analysis was used to assess the relationship of monocyte-to-lymphocyte ratio and neutrophil-to-lymphocyte ratio with persistent critical illness. Receiver operating characteristic curves and the Youden index were used to evaluate the discriminatory threshold of persistent critical illness. RESULTS: A total of 851 eligible patients were enrolled in the study: 328 patients with persistent critical illness and 523 without. The median levels of maximum neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio during intensive care unit stay were all higher in patients with persistent critical illness than in those without (11.46 vs. 9.13, p < .001 and 0.62 vs. 0.46, p < .001). Multivariate analysis revealed that the second (≥0.385, <0.693) and third (≥0.693) maximum monocyte-to-lymphocyte ratio tertiles were significantly associated with persistent critical illness after adjusting for confounding factors (odds ratio: 1.89, 95% confidence interval: 1.10-3.26, p = .021 and odds ratio 2.69, 95% confidence interval: 1.44-5.02, p = .002, respectively), whereas maximum neutrophil-to-lymphocyte ratio was not significantly correlated with persistent critical illness. The area under the curve for the maximum monocyte-to-lymphocyte ratio was 0.63 (95% confidence interval: 0.59-0.67), and the optimal cutoff was 0.65 for persistent critical illness. CONCLUSION: A high maximum monocyte-to-lymphocyte ratio during intensive care unit stay was independently related to persistent critical illness following severe trauma, although with limited sensitivity and specificity.

Mouse Model of Critical Persistent Inflammation, Immunosuppression, and Catabolism Syndrome.

ABSTRACT: Persistent inflammation, immunosuppression, and catabolism syndrome (PIICS) is a growing challenge in intensive care units (ICUs). PIICS causes a severe illness with high mortality. Currently, treatment is expensive, and the outcomes are dismal. Herein, we established a PIICS model to study the disease pathophysiology and its potential treatment. Using a modified sublethal cecal ligation and puncture (CLP) to induce sepsis (day 1) and the injection of lipopolysaccharide (LPS) to induce an aggravated inflammation response (day 11), CLP + LPS mice recapitulating PIICS features were successfully generated (day 14). Adult male mice were divided into CLP + LPS, CLP + daily chronic stress (DCS), CLP, DCS, LPS, and sham control groups. A survival curve was generated, and phenotypes were analyzed using markers for catabolism, inflammation, and immunosuppression. The CLP + LPS model showed two mortality peaks (after CLP and after LPS), whereas the CLP + DCS and CLP groups showed one peak. Surviving CLP + LPS mice exhibited significantly increased catabolism and inflammatory cytokine levels and aggravated inflammation, including organ inflammation. CLP + LPS mice exhibited strong immune suppression as evidenced by decreased splenic cluster of differentiation (CD)8+ and interferon-γ+CD8+ T cell counts and a concomitant and significant increase in the myeloid-derived suppressor cell population. This CLP+LPS-induced PIICS model differs from acute sepsis models, showing two mortality peaks and a protracted course of 14 days. Compared to previous PIICS models, ours shows a re-aggravated status and higher catabolism, inflammation, and immunosuppression levels. Our aim was to use the PIICS model to simulate PIICS pathophysiology and course in the ICU, enabling investigation of its mechanism and treatment.

[Analysis of risk factors of polymyxin B-associated acute kidney injury in intensive care unit patients with severe infection].

OBJECTIVE: To investigate the incidence and risk factors of polymyxin B-associated acute kidney injury (AKI) in patients with severe infections caused by extensive drug resistance Gram negative bacteria (XDR-GNB) in intensive care unit (ICU). METHODS: A retrospective study of adult patients with severe infection who received polymyxin B for more than 3 days in the department of critical care medicine of Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School from April 1st 2018 to January 31st 2020 were performed. AKI was diagnosed by Kidney Disease: Improving Global Outcomes (KDIGO) criteria. The baseline data, indicators during treatment period and prognostic factors were compared between AKI group and non-AKI group. Factors with statistically significant difference in univariate analysis and important clinical factors were included in the Logistic regression model to analyze the risk factors of AKI. RESULTS: Seventy-two patients were treated with polymyxin B for more than 3 days. Forty-nine patients were finally enrolled, with 32 patients developing polymyxin B-associated AKI, and the incidence was 44.4%. The baseline data was balanced in AKI group and non-AKI group, and there was no significant difference in the prognosis [death or discharge without medial order (cases): 14 vs. 6, discharged for improvement (cases): 18 vs. 11, χ2 = 0.329, P = 0.566]. Polymyxin B-associated AKI occurred from 1 day to 14 days after treatment, with an average of (6.8±3.8) days. Among the 32 AKI patients, 2 cases were lost to follow up after discharge, while renal function recovered in 18 cases and unrecovered in 12 cases. The prognosis of patients without recovery of renal function was significantly worse than that of patients with renal function recovery [death or discharge without medial order (cases): 12 vs. 2, discharged for improvement (cases): 0 vs. 16, P = 0.000]. Single factor analysis showed that daily dosage of polymyxin B in AKI group was higher than that in non-AKI group (mg: 151.6±23.7 vs. 132.4±30.3), numbers of patients with daily polymyxin B dose ≥ 150 mg, using vasoactive drugs, or severe hypoalbuminemia (albumin ≤ 25 g/L) were higher than those in non-AKI group (cases: 29 vs. 10, 18 vs. 4, 9 vs. 0), with statistically significant differences (all P < 0.05). Multivariate Logistic regression analysis showed that daily dosage of polymyxin B ≥ 150 mg and use of vasoactive drugs were independent risk factors for polymyxin B-associated AKI [odds ratio (OR) = 37.466, 95% confidence interval (95%CI) was 2.676-524.586, P = 0.007; OR = 22.960, 95%CI was 1.710-308.235, P = 0.018]. CONCLUSIONS: Comparing with non-AKI patients, more patients with polymyxin B-associated AKI had severe hypoalbuminemia, and the probability of using vasoactive drugs and the daily dose of polymyxin B were higher than non-AKI patients. Daily dose of polymyxin B ≥ 150 mg and using vasoactive drugs were independent risk factors for polymyxin B-associated AKI.

Metagenomic next-generation sequencing in the diagnosis of severe pneumonias caused by Chlamydia psittaci.

PURPOSE: Chlamydia psittaci infection in humans can lead to serious clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, rarely, death. Implementation of metagenomic next-generation sequencing (mNGS) gives a promising new tool for diagnosis. The clinical spectrum of severe psittacosis pneumonia is described to provide physicians with a better understanding and to highlight the rarity and severity of severe psittacosis pneumonia. METHODS: Nine cases of severe psittacosis pneumonia were diagnosed using mNGS. Retrospective analysis of the data on disease progression, new diagnosis tool, treatments, and outcomes, and the findings were summarised. RESULTS: Frequent symptoms included chills and remittent fever (100%), cough and hypodynamia (100%), and headache and myalgia (77.8%). All patients were severe psittacosis pneumonia developed respiratory failure, accompanied by sepsis in 6/9 patients. mNGS takes 48-72 h to provide the results, and help to identify diagnosis of psittacosis. Laboratory data showed normal or slightly increased leucocytes, neutrophils, and procalcitonin but high C-reactive protein levels. Computed tomography revealed air-space consolidation and ground-glass opacity, which began in the upper lobe of one lung, and spread to both lungs, along with miliary, nodular, or consolidated shadows. One patient died because of secondary infection with Klebsiella pneumoniae, while the other eight patients experienced complete recoveries. CONCLUSIONS: The use of mNGS can improve accuracy and reduce the delay in diagnosis of psittacosis. Severe psittacosis pneumonia responds well to the timely use of appropriate antibiotics.

Expression and significance of caveolin-1 in hepatitis B virus-associated hepatocellular carcinoma.

Caveolin-1 (Cav-1) is a major component of caveolae and has been recently identified as a tumor suppressor. As little is known about Cav-1 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), the aim of the present study was to investigate the expression and significance of Cav-1 in HBV-associated HCC. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the mRNA expression level of Cav-1 in 40 cases of HBV-associated HCC, the corresponding 11 non-tumor cases of HBV-associated chronic hepatitis, 29 non-tumor cases of HBV-associated cirrhosis and 6 cases of normal liver tissues. Immunohistochemical analysis indicated the expression of Cav-1, cluster of differentiation 34 and vascular endothelial growth factor (VEGF) in HBV-associated HCC tissue samples. In addition, the association of Cav-1 expression with angiogenesis and clinicopathological characteristics of HBV-associated HCC was also analyzed. RT-PCR results demonstrated that the expression rate of Cav-1 mRNA in HBV-associated HCC, non-tumor HBV-associated chronic hepatitis and cirrhosis liver tissues and control normal liver tissues from patients with metastatic carcinoma was 92.5, 85.0 and 16.7%, respectively. mRNA expression level of Cav-1 was significantly increased in chronic hepatitis, cirrhosis and HBV-associated HCC livers compared with normal control livers (P<0.05 and P<0.01, respectively). Cav-1 protein was detected by immunohistochemistry in 80% of the samples of HBV-associated HCC. Furthermore, Cav-1 and VEGF protein expression levels were correlated with microvessel density (MVD; γs<0.46, P=0.01 and γs<0.31, P=0.05, respectively). In addition, Cav-1 expression and MVD were significantly associated with metastasis (P=0.031 and P=0.046, respectively). In conclusion, Cav-1 may have an important role in the carcinogenesis and progression of HBV-associated HCC and angiogenesis may be affected by Cav-1 during this process.

[Construction of a novel bioreactor of bioartificial liver system with human bone marrow mesenchymal stem cells].

OBJECTIVE: To design a novel bioreactor of bioartificial liver system by using expanded and differentiated human bone marrow mesenchymal stem cells (hBMMSCs) as active cells. METHODS: hBMMSCs were isolated from bone marrow of volunteers and grown to 10(7) population, and then replanted into hollow fiber cartridge to expand continuously for 10 days. They were incubated in differentiation medium containing recombinant human hepatocyte growth factor (rhHGF), recombinant human fibroblast growth factor 4 (rhFGF-4), recombinant human oncostatin M (rhOSM), and the cells were induced to differentiate into hepatocyte-like cells for 21 days. The functions of the differentiated cells, such as synthesis of albumin (Alb), alpha fetoprotein (AFP) were determined. Eighteen days later, the functions of metabolism of ammonia and benzodiazepines, and synthesis of urea were monitored. The cellular synthesis rate of Alb was measured with flow cytometer. The glucose levels in the medium were measured during entire culture process. RESULTS: (1)Glucose-uptake in the cartridge was increased during the culture period, and at the end of culture, the number of cells in the cartridge increased to 10(9). (2)After induction, AFP was detected on day 6, reaching the peak level on day 12. Alb was detected on day 9. Eighteen days after being induced, the clearance rate of ammonia and benzodiazepines in the cartridge was 2.0-2.7 mmol/24 hours and 3.2-3.8 mg/24 hours, respectively, and urea production rate was 1.8-2.2 mmol/24 hours. (3)At the end of the culture, 66.18%-76.91% of the cells showed positive Alb expression. CONCLUSION: hBMMSCs can be multiply to construct a novel bioreactor of bioartificial liver system in a hollow fiber cartridge.

Optimization of an effective directed differentiation medium for differentiating mouse bone marrow mesenchymal stem cells into hepatocytes in vitro.

We have used a uniform design to explore the most effective directed differentiation medium (MEDDM) for differentiating mouse bone marrow mesenchymal stem cells (mMSCs) into hepatocytes. Our methods involved arranging eight differentiation medium groups following uniform design. Flow cytometry was used to evaluate the percentage of ALB+ and CK18+ cells in each group. Factors and their concentrations in the MEDDMs were then identified. The MEDDMs were evaluated by their ability to differentiate mMSCs into hepatocytes by RNA and protein expressions and synthesis functions. FGF at 35 ng/ml and OSM at 30 ng/ml in the medium yielded the highest percentage of ALB+ and CK18+ cells. During directed differentiation using MEDDMs, ALB, CK18, TTR, AFP mRNAs were expressed. ALB and CK18 proteins were detected in the cells. The differentiated cells produced albumin and urea in a time dependent manner. Uniform design was adequate for choosing the MEDDM of mMSCs. MEDDM containing 35 ng/ml FGF and 30 ng/ml OSM was effective in differentiating mMSCs into hepatocytes.

Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells.

AIM: The role of caveolin-1 (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program. RESULTS: RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L). CONCLUSION: NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti-angiogenesis therapy.

In vitro differentiation of mouse bone marrow mononuclear cells into hepatocyte-like cells.

AIM: : It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS: : Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS: : Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS: : BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.

Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro.

AIM: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. METHODS: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentiation of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. RESULTS: Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3beta (HNF-3beta, albumin (ALB), cytokeratin 18 (CK18), transthyretin (TTR), glucose-6-phosphate (G-6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3? ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. CONCLUSION: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

[Pharmacokinetics of vancomycin in continuous veno-venous hemofiltration].

OBJECTIVE: To investigate the pharmacokinetics of vancomycin in continuous veno-venous hemofiltration(CVVH) in order to determine appropriate vancomycin dosing strategies for patients receiving CVVH. METHODS: The serum concentrations of vancomycin were measured by TDx and the pharmacokinetics parameters were calculated. RESULTS: The pharmacokinetics of vancomycin during CVVH was fitted with open two-compartment model. At the beginning of CVVH, the pharmacokinetic parameters were: maximum plasma concentration (Cmax)=22.18 mg/L, minimum plasma concentration attained (Cmin)=5.82 mg/L, half-life of drug (T1/2)=5.75 h, apparent volume of distribution (Vd)=21.92 L, total body clearance of drug(CL)=3.49 L/h. On the 16 d of CVVH, the pharmacokinetic parameters were Cmax=38.70 mg/L, Cmin=16.50 mg/L, T1/2=33.32 h, Vd=12.92 L, CL=0.38 L/h. CONCLUSION: CVVH can significantly augment the clearance of vancomycin in acute renal failure patients. Dosing strategies for individualization of vancomycin therapy in patients receiving CVVH are proposed. Monitoring the serum concentration during CVVH is necessary.