Isolation of High Purity Mouse Mesenchymal Stem Cells through Depleting Macrophages Using Liposomal Clodronate

BACKGROUND@#The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion. @*METHODS@#In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate.This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes). @*RESULTS@#Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs.This method allowed the generation of numerous high-purity Sca-1 ＋ CD44 ＋ F4/80 - mBMSCs ([ 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays. @*CONCLUSION@#Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.

CCR5 deficiency in aged mice causes a decrease in bone mass

The CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that regulates chemotaxis and effector functions of immune cells. It also serves as the major co-receptor for the entry of human immunodeficiency virus (HIV). Recently, CCR5 inhibitors have been developed and used for the treatment or prevention of HIV infections. Additionally, it has been identified that CCR5 controls bone homeostasis by regulating osteoclastogenesis and the communication between osteoblasts and osteoclasts. However, the effects of CCR5 inhibition on bone tissue in elderly patients are unknown. This study aimed to examine the bone phenotype of aged CCR5 knockout (KO) mice. Femoral and tibial bones were isolated from 12-month and 18-month old wild-type (WT) and CCR5 KO mice, and microcomputed tomography and histology analyses were performed. Twelve-month-old CCR5 KO mice exhibited a decreased trabecular bone mass and cortical bone thickness in both femoral and tibial bones compared with age-matched WT mice. Eighteen-month-old mice also showed a decreased trabecular bone mass in femurs compared with control WT mice, but not in tibial bones. Unlike in 12-month-old mice, the cortical margin of femurs and tibias in 18-month-old mice were rough, likely because they were aggravated by the deficiency of CCR5. Overall, our data suggest that the deficiency of CCR5 with aging can cause severe bone loss. When CCR5 inhibitors or CCR5 inactivating technologies are used in elderly patients, a preventive strategy for bone loss should be considered.

Quantitative Analysis of Tooth Mineral Content by High Resolution Micro-computed Tomography

Teeth and bones are highly mineralized tissues containing inorganic minerals such as calcium phosphate, and a growing number of evidences show that their mineral content is associated with many diseases. Although the quantification of mineral contents by micro-computed tomography(micro- CT) has been used in diagnosis and evaluation for treating bone diseases, its application for teeth diseases has not been well established. In this study, we attempted to estimate a usefulness of a high-resolution micro-CT in analysis of human teeth. The teeth were scanned by using the Skyscan 1172 micro-CT. In order to measure tooth mineral content, beam hardening effect of the machine was corrected with a radiopaque iodinecontaining substance, iodoacetamide. Under the maximum resolution of 6.6 µm, X-ray densities in teeth and hydroxyapatite standards were obtained with Hounsfield unit (HU), and they were then converted to an absolute mineral concentration by a CT Analyzer software. In enamel layer of cusp area, the mean mineral concentration was about 2.14 mg/mm³ and there was a constant mineral concentration gradient from the enamel surface to the dentinoenamel junction. In the dentin of middle 1/3 of tooth, the mean mineral concentration was approximately 1.27 mg/mm³ and there was a constant mineral concentration gradient from the outer of root to the pulp side, ranging from 1.3 to 1.06 mg/mm³. In decay region of dentin, the mineral content was gradually decreased from the intact inner side to the decayed surface. These results suggest that high-resolution micro-CT can be as a useful tool for non-invasive measurement of mineral concentration in teeth.

Causes of Fever in the ICU - A Prospective, Cohort Study / 대한구급학회지

BACKGROUND: Fever develops in 70% of ICU patients. In the present study, we tried to figure out causes of fever and the prognosis of febrile patients in the ICU in a prospective, cohort method. METHODS: From February to June 2007, patients admitted to medical ICU were daily screened and those who developed fever were enrolled. 237 consecutive admissions of 237 patients over a 5-month period were analyzed. Clinical parameters, including demographic data, underlying diseases, duration of ICU stay, causes of fever and final outcome were analyzed. RESULTS: Fever (core temperature > or =38.3degrees C) was present in 8% of admission, and it was caused by infective (84.2%) and non-infective processes (15.8%). Most fever occurred within first 5 days in the course of the admission (68.4%) and most lasted less than 5 days (57.9%). The median Acute Physiology and Chronic Health Evaluation (APACHE) III score at the time of fever was 43 (+/-19). Those with infectious fever had no significant differences in terms of severity of diseases in comparison with those with non-infectious cause of fever. The most common cause of infective fever was pneumonia (n=11). Prolonged fever (> or =5 days), all of which was caused by infection, occurred in 11 patients. Those with prolonged fever had higher mortality rate than short duration of fever (37.5% vs 0%, p<0.05). CONCLUSION: Infection, especially pneumonia is common cause of fever in the ICU. Prolonged fever is associated with high mortality rate.

Multiplex PCR of Endotracheal Aspirate for the Detection of Pathogens in Ventilator Associated Pneumonia / 결핵및호흡기질환

BACKGROUND: Early identification of pathogens can improve the prognosis of patients with ventilator associated pneumonia (VAP). In the present study, we evaluated the feasibility of performing multiplex PCR for endotracheal aspirates to detect three important pathogens (P. aeruginosa, K. pneumoniae and MRSA) in patients with VAP. METHODS: The endotracheal aspirates of 24 patients were collected within 24 hours of the diagnosis of VAP for performing multiplex PCR. Forward and reverse primers were designed to target the specific site of each pathogen (the oprL gene for P. aeruginosa, 16S rRNA for K. pneumoniae and the mec gene for MRSA). We analyzed the clinical data of the VAP patients, including the culture reports for the endotracheal aspirates. RESULTS: Twenty-four patients (M:F=18:6, mean age=70+/-11) with VAP were enrolled. Pathogens were isolated from 11 patients (P. aeruginosa in 2, K. pneumoniae in 1, MRSA in 2, other enteric Gram negative bacilli in 3, S. pneumoniae in 2 and mixed infection in 1). Multiplex PCR detected three cases of P.aeruginosa (2 cases coincided with the culture reports) and four cases of K. pneumoniae (1 matched with the culture report). PCR detected two MRSA cases, which did not coincide with the culture reports. CONCLUSION: Multiplex PCR of the endotracheal aspirate showed some ability to detect Gram negative bacilli, although caution is required when interpreting the results.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Augments Acute Lung Injury via Its Neutrophil Priming Effects

Granulocyte macrophage-colony stimulating factor (GM-CSF) has immuno-stimulatory effects. We hypothesized that GM-CSF plays an important role both in lipopolysaccharide (LPS)- and hemorrhage-induced acute lung injury (ALI). We also postulated that GM-CSF augments LPS-induced inflammation by priming neutrophils. ALI was induced in GM-CSF-/- or control C57BL mice either by LPS injection or by hemorrhage. Lung inflammation (by lung expression for tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), interleukin-1beta (IL-1beta), interleukin- 6 (IL-6), and keratinocyte-derived chemokine) and lung injury (by myeloperoxidase and evans blue dye assay) were evaluated after ALI. Incremental doses of LPS (0, 1, 10, and 100 ng/mL) and GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils. The expression of TNF-alpha, MIP-2, and IL-1beta was evaluated with enzyme linked immunosorbent assay. The mRNA expression of three cytokines, and the nuclear translocation of nuclear factor kappa B (NF kappa-B) were evaluated by reverse transcriptase-polymerase chain reaction and electropnoretic mobility shift assay, respectively. GM-CSF -/- mice showed decreased neutrophil infiltration, less leakage, and lower expression of cytokines in the lung after LPS or hemorrhage. GM-CSF augmented LPS-induced protein and mRNA expression of TNF-alpha, MIP-2 and IL-1beta, which was mediated by increased intra-nuclear translocation of NF-kappa B. GM-CSF plays an important role in high-dose LPS and hemorrhage-induced ALI, which appears to be mediated by its priming effect on neutrophils.

A case of hypersensitivity vasculitis after influenza vaccination / 대한내과학회지

No abstract available.

A Case of Empyema and Mediastinitis by Non-typhi Salmonella / 결핵및호흡기질환

There are few reports of the pleuropulmonary involvement of a non-typhi Salmonella infection in immunocompromised patients with AIDS, malignancy, collagen vascular diseases, extended use of corticosteroids, sickle cell disease, or diabetes. We report a case of a non-immunocompromised patient who presented with concomitant empyema and mediastinitis due to Salmonella without a comorbid disease. A 26-year-old male patient, with a history of pneumonia 5 years earlier and having lived abroad for several years, presented chronic cough and febrile sensation. Pneumonia, empyema and mediastinitis were noted in a chest CT scan and Salmonella enteritidis and beta-hemolytic streptococcus were identified from a culture of the pleural fluid. Initially, he was treated with cefepime, metronidazole and clarithromycin. He was cured clinically and radiographically after an 8 week treatment with antibiotics. In conclusion, this report suggests that S. enteritidis can cause empyema and mediastinitis, albeit rarely.

Diagnostic Value of ADA Multiplied by Lymphocyte to Neutrophil Ratio in Tuberculous Pleurisy / 결핵및호흡기질환

BACKGROUND: Many diagnostic approaches for defining the definitive cause of pleurisy should be included due to the large variety of diseases resulting in pleural effusion. Although ADA is a useful diagnostic tool for making a differential diagnosis of pleural effusion, particularly for tuberculous pleural effusion, a definitive diagnostic cut-off value remains problematic in Korea. It was hypothesized that ADA multiplied by the Lymphocyte/Neutrophil ratio(L/N ratio) might be more powerful for making a differential diagnosis of pleural effusion. METHODS: One hundred and ninety patients, who underwent thoracentesis and treatment in Chung-Ang University Hospital from January, 2005 through to February 2006, were evaluated. The clinical characteristics, radiologic data and the examination of the pleural effusion were analyzed retrospectively. RESULTS: 1. Among the 190 patients, 59 patients (31.1%) were diagnosed with tuberculous pleurisy, 45 patients(23.7%) with parapneumonic effusion, 42 patients(22.1%) with malignant effusions, 36 patients(18.9%) with transudate, and 8 patients(4.2%) with empyema. One hundred and twenty one patients were found to have an ADA activity of 1 to 39 IU/L(63.7%). Twenty-nine were found to have an ADA activity of 40 to 75 IU/L(15.3%) and 40 were found to have an ADA activity of 75 IU/L or greater(21.0%). 2. Among the patients with tuberculous pleurisy, 5(8%), 18(30%) and 36 patients(60%) had an ADA activity ranging from 1 to 39 IU/L, 40 to 75 IU/L, and 75 IU/L or greater, respectively. In those with an ADA activitiy 40 to 75 IU/L, 18 patients(62%) had tuberculous pleurisy, 9(31%) had parapneumonic effusion and empyema, and 1(3.4%) had a malignant effusion. 3. In those with an ADA activity of 40 to 75 IU/L, there was no significant difference between tuberculous pleurisy and non-tuberculous pleural effusion(tuberculous pleurisy : 61.3 +/- 9.2 IU/L, non-tuberculous pleural effusion : 53.3+/-10.5 IU/L). 4. The mean L/N ratio of those with tuberculous pleurisy was 39.1 +/- 44.6, which was significantly higher than non- tuberculous pleural effusion patients (p<0.05). The mean ADA x L/N ratio of the tuberculous pleurisy patients was 2,445.7 +/- 2,818.5, which was significantly higher than the non-tuberculous pleural effusion patients (level p<0.05).5. ROC analysis showed that the ADA x L/N ratio had a higher diagnostic value than the ADA alone in the group with an ADA between 40-75 IU/L. CONCLUSION: The ADA multiplied by the lymphocyte-to-neutrophil ratio might provide a more definitive diagnosis of tuberculous pleurisy.

Proinflammatory Effects of High Mobility Group B1 (HMGB1) Versus LPS and the Mechanism of IL-8 Promoter Stimulation by HMGB1 / 결핵및호흡기질환

BACKGROUND: High mobility group box 1 (HMGB1) is a novel, late mediator of inflammation. This study compared the pro-inflammatory effects of LPS and HMGB1. The transcriptional factors that play an important role in mediating the HMGB1-induced stimulation of IL-8 were also evaluated. METHODS: RAW264.7 cells were stimulated with either LPS (100 ng/ml) or HMGB1 (500 ng/ml). The TNF-alpha, MIP-2 and IL-1beta levels in the supernatant were evaluated by ELISA at 0, 2, 4, 8, 12 and 24h after stimulation. An acute lung injury was induced by an injection of LPS (5 mg/kg) or HMGB1 (2.5 mg/kg) into the peritoneum of the Balb/c mice. The lung cytokines and MPO activity were measured at 4h (for LPS) or 24h (for HMGB1) after the injection. The transcriptional factor binding sites for NF-IL6, NF-kappaB and AP-1 in the IL-8 promoter region were artificially mutated. Each mutant was ligated with pIL-6luc and transfected into the RAW264.7 cells. One hour after stimulation with HMGB1 (500 ng/ml), the cell lysate was analyzed for the luciferase activity. RESULTS: The expression of MIP-2, which peaked at 8h with LPS stimulation, increased sequentially until 24h after HMGB1 stimulation. An intraperitoneal injection of HMGB1, which induced a minimal increased in IL-1beta expression, provoked the accumulation of neutrophils the lung. A mutation of AP-1 as well as NF-kappaB in the IL-8 promoter region resulted in a lower luciferase activity after HMGB1 stimulation. CONCLUSION: The proinflammatory effects of HMGB1, particularly on IL-8, are mediated by both NF-kappaB and AP-1.

Microenvironments and Cellular Proliferation Affected by Oxygen Concentration in Non-Small Cell Lung Cancer Cell Line / 결핵및호흡기질환

BACKGROUND: Abnormal angiogenesis can induce hypoxia within a highly proliferating tumor mass, and these hypoxic conditions can in turn create clinical problems, such as resistance to chemotherapy. However, the mechanism by which hypoxia induces these changes has not yet been determined. Therefore, this study was conducted to determine how hypoxia induces changes in cell viability and extracellular microenvironments in an in vitro culture system using non-small cell lung cancer cells. METHODS: The non-small cell lung cancer cell line, A549 was cultured in DMEM or RPMI-1640 media that contained fetal bovine serum. A decrease in the oxygen tension of the media that contained the culture was then induced in a hypoxia microchamber using a CO2-N2 gas mixture. A gas analysis and an MTT assay were then conducted. RESULTS: (1) The decrease in oxygen tension was checked the anaerobic gas mixture for 30 min and then reoxygenation was induced by adding a 5% CO2-room air gas mixture to the chamber. (2) Purging with the anaerobic gas mixture was found to decrease the further oxygen tension of cell culture media. (3) The low oxygen tension resulted in a low pH, lactic acidosis and a decreased glucose concentration in the media. (4) The decrease in glucose concentration that was observed as a result of hypoxia was markedly different when different types of media were evaluated. (5) The decrease in oxygen tension inhibited proliferation of A549 cells. CONCLUSION: These data suggests that tumor hypoxia is associated with acidosis and hypoglycemia, which have been implicated in the development of resistance to chemotherapy and radiotherapy.

A Case of Pseudomembranous Necrotizing Bronchial Aspergillosis in An Old Age Host / 결핵및호흡기질환

Invasive aspergillus tracheobronchitis is uncommon manifestation of infection due to Aspergillus species, occurring in severely immunocompromised patients who are generally neutropenic with haematological diseases, AIDS, or after heart and lung transplantation. The pseudomembranous form is the most severe condition and is usually fatal despite treatment with antifungal agents. However, there are a few cases reported with no apparent severe compromise in the host defences. We encountered a pseudomembranous necrotizing bronchial aspergillosis in a 73-year old male patient, who was treated successfully with antifungal agents.

Risk Factors for Mortality in Community-Acquired Pneumonia Patients Admitted to a Referral Hospital / 결핵및호흡기질환

BACKGROUND: Pneumonia is the most common cause of death among infectious diseases with community-acquired pneumonia being the sixth leading cause of death in the USA. In Korea, several studies have evaluated the prognosis of community-acquired pneumonia with a limited number of patients and risk factors. This study, evaluated all the possible risk factors (including the pneumonia severity index; PSI) in for the community-acquired pneumonia patients admitted to a referral hospital. METHODS: The medical records of patients admitted to the Chung-Aug University Yongsan Hospital between January 2002 and January 2005 for community-acquired pneumonia were reviewed retrospectively. The demographic data, comorbidity, radiographic findings and laboratory results which might influence the prognosis of pneumonia were analyzed. RESULTS: Among 179 patients admitted for community-acquired pneumonia, 29 patients died (mortality 16%). The risk factors for mortality in the comorbidity category were congestive heart failure and a myocardial infarction. The laboratory data, showed that albumin, LDH, total cholesterol, HDL, PT, aPTT, hemoglobin and blood urea nitrogen (BUN) were related to the prognosis. For the pneumonia severity index, the mortality rate increased in a step-wise manner from class I through class V. CONCLUSIONS: Comorbidities such as congestive heart failure and myocardial infarction as well as the albumin, LDH, total cholesterol, HDL cholestreol, prothrombin time, activated partial thrombotin time, hemoglobin and blood urea nitrogen(BUN) are important risk factors for mortality in patients with community-acquired pneumonia. PSI is a valuable index for evaluating the prognosis of community-acquired pneumonia.

The Clinical Manifestation of Pulmonary Infection in AIDS Patients / 결핵및호흡기질환

BACKGROUND: In AIDS patients, the respiratory tract is one of the most frequently involved site of by an opportunistic infection, and an even common, casual pulmonary infection manifests in a peculiar ways in AIDS patients. In Korea, because of the low prevalence of AIDS, there is insufficient data compared with other Southeast Asian counties. However, considering the sexual behavior of the younger generation, it is expected that it will not be long before AIDS becomes a major public health issue in Korea. This study examined the clinical manifestation of HIV positive people and AIDS patients. The pulmonary manifestion of HIV-positive was evaluated. This study focused on the differences in the clinical manifestation between AIDS patients with pulmonary disease and simple HIV positive people. The characteristics of common pulmonary infections in AIDS patients were also analyzed. METHOD: The medical records of 28 HIV positive patients who visited the hospital of ChungAng University Hospital from January, 2001 to February, 2006 were analyzed retrospectively. RESULT: Twenty-seven patients out of 28 HIV positive patients were male and the average age was 40.6(23-65). Patients in their thirties were most commonly affected. Elven patients had pulmonary diseases. Pulmonary tuberculosis (4 cases) and pneumocystis pneumonia (4 cases) were the most common respiratory infection. One patient showed a peculiar type of systemic cryptococcus. which was accompanied by lung and pleural dissemination. The CD4+lymphocyte count of patients with a pulmonary infection was significantly lower in patients with a pulmonary manifestation than those with only a HIV infection (79.5/mm3 vs 400/mm3, respectively)(p<.05). Patients with pulmonary disease were in a more severe immunosuppressive state. There were 4 patients with pulmonary tuberculosis, 4 with pneumocystis pneumonia, 4 with secondary syphilis, 2 with primary syphilis, and 2 with HZV infection. The average CD4 lymphocyte counts was 56/mm3 in those with pulmonary tuberculosis, 42/mm3 in those with pneumocystis pneumonia, and 455/mm3 in those with secondary syphilis. CONCLUSION: This study examined the clinical manifestation of HIV positive patients, particularly AIDS patients with pulmonary disease, A more severe immunosuppressive status was observed in HIV-related pulmonary compared with those with HIV-related extrapulmonary disease, and the frequency of pulmonary tuberculosis in pulmonary disease was higher than expected. Respiratory infection in AIDS manifest in uncommon ways e.g. disseminated cryptococcosis involving the lung and pleura. Evidently, AIDS patients with a respiratory infection have a more severe form of immunosuppression than those with a simple HIV infection. As expected, patients with a pulmonary infection were in a more severe immunosuppressed state than those with a simple HIV infection. Opportunistic infections can show peculiar clinical presentations in AIDS patients.