NLRP3 upregulation in A549 cells co-cultured with THP-1 macrophages under hypoxia via deregulated TGF-ß signaling.

NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is one of the key components of the inflammasome. NLRP3 also participates in the regulation of fibrosis independent of the inflammasome. In this study, we analyzed the mechanism of upregulation of NLRP3 expression in A549 cells co-cultured with THP-1 macrophages under hypoxia. Upregulation of NLRP3 was suppressed after treatment with inhibitors of TGF-ß receptor or p38, but not with inhibitors of the IL-1 receptor and SMAD3. The analysis of downstream molecules of TGF-ß signaling in A549 cells co-cultured with THP-1 macrophages under hypoxia showed that TGFBR1 was upregulated and SMAD7 was downregulated. Taken together, these results suggest that the upregulation of NLRP3 in A549 cells is associated with deregulated TGF-ß signaling and that the interaction between NLRP3 and TGF-ß signaling plays a fundamental role in fibrogenesis.

Upregulation of osteopontin expression via the interaction of macrophages and fibroblasts under IL-1b stimulation.

BACKGROUND: Fibrosis is attributed to dysregulation of tissue-remodeling. In remodeling areas, fibroblasts and macrophages actively make contact with each other. Osteopontin (OPN) is a pro-fibrotic molecule, whose expression is upregulated by interleukin (IL)-1ß via secretion of its downstream cytokines, such as IL-6. Here, we investigated the effect of interaction between fibroblasts and macrophages under IL-1ß stimulation on the expression of OPN. METHODS: We used human lung fibroblasts and THP-1 macrophages differentiated from THP-1 cells using phorbol 12-myristate 13-acetate. These cells were either cultured alone or co-cultured under IL-1ß stimulation. Secretion of OPN and IL-6 were examined by enzyme-linked immunosorbent assay, and mRNA expression was assessed by quantitative real-time PCR. The effects of siRNA against IL-6 or OPN on OPN expression were evaluated. RESULTS: OPN expression increased when fibroblasts and THP-1 macrophages were co-cultured under IL-1ß stimulation. The siRNA against IL-6 in fibroblasts suppressed the upregulation of OPN expression during co-culture, whereas siRNA against IL-6 in THP-1 macrophages did not. The upregulation of expression of OPN mRNA in fibroblasts or THP-1 macrophages when co-cultured under IL-1ß stimulation was mediated by IL-6 from fibroblasts. OPN from THP-1 macrophages was involved in the increase of OPN expression in fibroblasts. CONCLUSIONS: The present study revealed the crosstalk between fibroblasts and THP-1 macrophages under IL-1ß stimulation, where IL-6 from fibroblasts, stimulated by IL-1ß, upregulated OPN expression in fibroblasts themselves via increase in OPN from THP-1 macrophages. The fibroblasts/macrophages network may induce activation or qualitative changes in both cells, which contributes to inflammation-associated fibrosis.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

BACKGROUND: ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. METHODS: We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. RESULTS: Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. CONCLUSION: The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

IL-6 trans-signaling is another pathway to upregulate Osteopontin.

BACKGROUND: Osteopontin (OPN) is a pro-fibrotic molecule upregulated by pro-inflammatory cytokines. Interleukin (IL)-6 functions downstream of IL-1ß and has unique signal pathways: classic- or trans-signaling via membrane-bound IL-6R or soluble IL-6R (sIL-6R). We investigated the effect of IL-6 trans-signaling on the upregulation of OPN. METHODS: We used THP-1 cells and THP-1 macrophages differentiated from THP-1 cells using phorbol 12-myristate 13-acetate (PMA). After IL-1ß stimulation, expression of OPN, IL-6, sIL-6R, and a disintegrin and metalloproteinase 17 (ADAM17) was examined by ELISA and quantitative PCR. The effects of anti-human IL-6 neutralizing antibody, soluble gp130 (sgp130, IL-6 trans-signaling-specific inhibitor), TAPI-1 (ADAM inhibitor) and siRNA against IL-6R or ADAM17 on OPN expression were evaluated. RESULTS: IL-1ß increased OPN and induced IL-6 in THP-1 macrophages. Anti-IL-6 neutralizing antibody and siRNA against IL-6R inhibited OPN upregulation induced by IL-1ß. TAPI-1 significantly inhibited the increase in sIL-6R induced by IL-1ß. Treatment with sgp130 attenuated OPN elevation by IL-1ß, whereas sgp130 did not change OPN levels in THP-1 macrophages without IL-1ß stimulation. ADAM17 was expressed in THP-1 macrophages and THP-1 cells and IL-1ß stimulation significantly increased ADAM17 expression, regardless of PMA treatment. TAPI-1 and siRNA against ADAM17 significantly inhibited OPN increased by IL-1ß. CONCLUSIONS: IL-6 and sIL-6R induced by IL-1ß may trigger IL-6 trans-signaling, contributing to the upregulation of OPN in THP-1 macrophages. Macrophages may be used as a source of IL-6 and sIL-6R and evoke IL-6 trans-signaling.

A Novel Mutation in the Fibrinogen Bß Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia.

We found a novel heterozygous mutation in the fibrinogen Bß chain (c.490G>A) of a 3-year-old girl with congenital hypofibrinogenemia. To clarify the complex genetic mechanism, we made a mini-gene including a FGB c.490G>A mutation region, transfected it into a Chinese Hamster Ovary (CHO) cell line, and analyzed reverse transcription (RT) products. The assembly process and secretion were examined using recombinant mutant fibrinogen. Direct sequencing demonstrated that the mutant RT product was 99 bp longer than the wild-type product, and an extra 99 bases were derived from intron 3. In recombinant expression, a mutant Bß-chain was weakly detected in the transfected CHO cell line, and aberrant fibrinogen was secreted into culture media; however, an aberrant Bß-chain was not detected in plasma. Since the aberrant Bß-chain was catabolized faster in cells, the aberrant Bß-chain in a small amount of secreted fibrinogen may catabolize in the bloodstream. FGB c.490G>A indicated the activation of a cryptic splice site causing the insertion of 99 bp in intron 3. This splicing abnormality led to the production of a Bß-chain possessing 33 aberrant amino acids, including two Cys residues in the coiled-coil domain. Therefore, a splicing abnormality may cause impaired fibrinogen assembly and secretion.

[Reversed Clinicopathological Conference].

Routine laboratory tests are the most frequently performed among clinical laboratory tests, and they can provide important information for the diagnosis and treatment of patients. They are more useful when sev- eral data are combined to interpret the pathophysiological state of a patient. Changes of routine laboratory data are important even when they are within their reference ranges, and they sometimes show a more de- tailed condition of the patient. In this RCPC, we-focused on changes of albumin and electrolytes, bacterial infections, and anemia. [Review].

Three-dimensional analysis of alveolar wall destruction in the early stage of pulmonary emphysema.

The morphological mechanism of alveolar wall destruction during pulmonary emphysema has not been clarified. The aim of this study was to elucidate this process three-dimensionally. Lung specimens from five patients with pulmonary emphysema were used, and five controls with normal alveolar structure were also examined. Sections 150 µm thick were stained with hematoxylin and eosin, elastica, and silver impregnation, and immunostained with selected antibodies. We examined these sections three-dimensionally using a laser confocal microscope and a light microscope. There were only a few Kohn's pores and no fenestrae in the normal alveoli from the controls. In the lungs of the emphysema patients a small rupture appeared in the extremely thin alveolar wall among the alveolar capillaries. This rupture enlarged to form a circle surrounded by the capillaries, which was called an alveolar fenestra. Two neighboring fenestrae fused by breakdown of the collapsed or cord-like capillary between them to form a large fenestra. The large fenestrae fused repeatedly to become larger, and these were bordered by thick elastic fibers constructing an alveolar framework. Alveolar wall destruction during emphysema could start from small ruptures of the alveolar wall that become fenestrae surrounded by capillaries, which fuse repeatedly to become larger fenestrae rimmed with elastic fibers. The alveolar capillary network could initially prevent enlargement of the fenestrae, and the thick elastic fibers constituting the alveolar framework could secondarily prevent destruction of the alveolar wall structure.

[How to Interpret Data of Routine Laboratory Tests (Basic Laboratory Tests)--Various Methods to Interpret Routine Laboratory Data in 3 Clinical Laboratory Facilities--A Method at Shinshu University Hospital].

Routine laboratory tests are the most frequently performed among clinical laboratory tests, and they can provide important information for the diagnosis and treatment of patients. They are more useful when several data are combined to interpret the pathophysiological state of a patient. Changes of routine laboratory data are important even when they are within their reference ranges, and they sometimes show a more detailed condition of the patient. In this symposium, we demonstrated our method at Shinshu University Hospital, involving the evaluation of 13 conditions of the whole body or each organ by simultaneously interpreting some to several laboratory data.

[Identification of Clinical Thymidine-Dependent Small-Colony Variants of Staphylococcus aureus by Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry].

In this study, the performance of MALDI-TOF MS was evaluated for the identification of clinical thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus. We performed identification of a total of 15 S. aureus TD-SCVs by using biochemical tests, 16S rRNA gene sequencing and MALDI-TOF MS analysis. Although the biochemical method using MicroScan panels could not identify all isolates due to insufficient growth in the control well. MALDI Biotyper (Bruker Daltonics) could correctly identify all of them. Two sample preparation methods, the direct transfer-formic acid method and ethanol-formic acid method, for measurement by MALDI Biotyper made no difference in results. MALDI-TOF MS is useful identification of S. aureus TD-SCVs.

[A Case of Secondary Cryofibrinogenemia with Cholangiocarcinoma and Deep Venous Thrombosis].

Cryofibrinogen (CF) is a type of cryoprotein (CP) that can precipitate in cooled plasma but not in serum, and resolves upon warming. We identified a case of secondary cryofibrinogenemia with cholangiocarcinoma and deep venous thrombosis. The patient's cryocrit measured using a Wintrobe tube was 19% in sodium citrate plasma stored for 7 days at 4 degrees C. We performed quantitative analysis of plasma proteins (fibrinogen, IgG, IgA, IgM, C3, C4, α1-antitrypsin, and C-reactive protein) before and after precipitation for 12 hours at 4 degrees C. The plasma fibrinogen concentration decreased by 16.7% (120 mg/dL --> 100 mg/dL), whereas the others were unaffected by precipitation. The CP purified from the patient's plasma was washed three times with saline and subjected to Western blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analyses. Western blot analysis indicated that the purified CP was composed of not only fibrinogen but also fibronectin, α1-antitrypsin, α2-macroglobulin, coagulation factor VIII, and IgG, IgA, and IgM. Interestingly, SDS-PAGE analysis showed that the molecular weight of the patient's CF differed from that of purified normal fibrinogen (340 KDa) and consisted of several low-molecular-weight bands (50-250 KDa). From these results, we speculated that CF found in this case was a mixture of degradated fibrinogen and some plasma proteins. In summary, cryofibrinogenemia is a rare and under-recognized disease. Sample information in routine clinical practice is valuable to diagnose this disease.

[Abnormal Serum Total Protein Measurement by Lipoprotein-X in an Infant with Biliary Atresia].

Lipoprotein-X (LP-X) in cholestatic jaundice causes abnormal reaction in assays for low-density lipoprotein-cholesterol, but the effects on other test items are unknown. Here, we report an infant with biliary atresia showing abnormal reaction in total serum protein assay using the biuret method, and lipoprotein-X (LP-X) was then detected. In this 11-month-old female infant, jaundice was observed at 2 months old, and a diagnosis of biliary atresia was made. On biochemical tests at 12 months old, the total serum protein concentrations detected by the biuret method were very high, and the response curve and linearity of dilution were abnormal. LP-X was detected by agar electrophoresis. In addition and recovery experiments with normal serum fractionation of the patient's LP-X-rich lipoprotein fraction prepared by ultracentrifugation, normal γ-globulin fractionation showed an abnormal reaction by the biuret method. In infants with biliary atresia, we showed that the total serum protein assay by the biuret method was influenced by LP-X-rich lipoprotein, which may be caused by abnormal reaction of LP-X and γ-globulin. [Case Report].

Epithelial-mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions.

Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-ß1 and IL-1ß in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-ß1 inhibitor or IL-1ß neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-ß1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-ß1 in A549 cells and THP-1 macrophages and that of IL-1ß in THP-1 macrophages when each cells were co-cultured. Anti-IL-1ß neutralizing antibody attenuated TGF-ß1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1ß from THP-1 macrophages up-regulated the TGF-ß1 from A549 cells and THP-1 macrophages, and then the TGF-ß1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.

N-homocysteinylation of apolipoprotein A-I impairs the protein's antioxidant ability but not its cholesterol efflux capacity.

A high homocysteine (Hcy) level is a risk factor for atherosclerosis. Hcy can be added to proteins through a process known as N-homocysteinylation. This is thought to be a potential cause of atherosclerosis induction. We previously reported that N-homocysteinylated apolipoprotein A-I (N-Hcy-apoA-I) was identified in normal human plasma. In this study, the effect of N-homocysteinylation on the functions of apoA-I was examined. A kinetic study using dimyristoyl phosphatidylcholine (DMPC) liposomes indicated that N-Hcy-apoA-I showed increased lipid-binding activity compared to wild-type apoA-I. Two reconstituted high-density lipoprotein (rHDL) particles of different sizes (approximately 8.2 nm and 7.6 nm in diameter) were produced by mixing apoA-I and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). However, an increased ratio of large to small particles was found in rHDL prepared with N-Hcy-apoA-I. The normal apoA-I antioxidant ability, estimated by the suppression of conjugated diene formation in low-density lipoprotein (LDL) induced by copper sulfate oxidation, was considerably impaired when using N-Hcy-apoA-I. Although N-Hcy-apoA-I functioned as an oxidant, no significant difference was observed in the cholesterol efflux capacity from THP-1 macrophages between wild-type apoA-I and N-Hcy-apoA-I. These results suggest that N-Hcy-apoA-I might be proatherogenic due to its oxidative behavior but not an attenuation of cholesterol efflux capacity.

[Let's interpret laboratory data as physical findings].

It is an important role of a clinical laboratory to provide accurate results of examinations promptly when a physician requires them, and this may be generally achieved. What more can technologists do to improve the medical service? It is more helpful for other medical staff to report crude laboratory data with adequate comments, which can be easily and promptly used in medical treatment. In this reversed clinicopathological conference, the admission periods of patients were divided into several phases: the admission day, from the 1st to 7th days, and from the 1st to 15th days. We closely analyzed routine laboratory data in each phase, and commented on what might be useful for the medical staff to the grasp the pathological state of patients in each phase. If comments from the laboratory become commonly acceptable, clinical laboratories will come to play a more valuable role in medical practice.

Combination of white blood cell count and left shift level real-timely reflects a course of bacterial infection.

BACKGROUND: The efficacy of white blood cell (WBC) count and left shift in predicting bacterial infections has been controversial. The aim of this study was to prove that WBC count and left shift reflect a course of bacterial infection. MATERIALS: Six patients in whom the onset of bacterial infection had been determined and successful treatment had been done were selected. Manual 100-cell differential counts were repeated at least every 24 hr. RESULTS: WBC count and left shift divided a course of bacterial infection into five phases. In the first phase of bacterial infection (0-10 hr after the onset), WBC count decreased to fewer than reference range without left shift. In the second phase (about 10-20 hr), low WBC count continued and left shift appeared. In the third phase (one to some days), WBC count increased above reference range with left shift. In the fourth phase (some to several days), high WBC count continued without left shift. In the fifth phase, WBC count went down into reference range without left shift. CONCLUSIONS: A combination of WBC count and left shift real-timely reflected a course of bacterial infection from the onset to healing. And we could judge which bacterial infection is adequately treated or not only by the above two routine laboratory tests.

[16S rRNA gene sequence analysis for bacterial identification in the clinical laboratory].

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, and can lead to the recognition of novel pathogens and noncultured bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques is impossible or difficult. However, there are some cases in which 16S rRNA gene sequence analysis can not differentiate closely related bacteria such as Shigella spp. and Escherichia coli at the species level. Thus, it is important to understand the advantages and disadvantages of 16S rRNA gene sequence analysis.

Consumption of nonfat milk results in a less atherogenic lipoprotein profile: a pilot study.

BACKGROUND: An increase in plasma low-density lipoprotein (LDL) is a well-known risk factor in the development of atherosclerosis. Dairy consumption may lower the risk of atherosclerosis; however, studies on the effects of milk on cardiovascular risk factors are still scarce. We were interested in investigating whether the intake of milk improves the atherogenic lipoprotein profile. AIMS: We investigated the effects of consuming whole or nonfat milk on plasma lipoprotein composition in healthy Japanese subjects as a pilot study. METHODS: Normolipidemic subjects consumed 500 ml of whole milk (whole milk group; n=7) or nonfat milk (nonfat milk group; n=7) every day for 2 weeks. RESULTS: The consumption of nonfat milk resulted in a lowering of plasma triglyceride (TG) and phospholipid levels and TG level in high-density lipoprotein (HDL) and increased the plasma apolipoprotein (apo) C-III level. In addition, the TG/cholesterol ratios in HDL and LDL were significantly decreased, and LDL particles became larger. In contrast, the only changes observed following whole milk consumption were increases in the plasma levels of apoC-III and apoE. CONCLUSIONS: These findings suggest that consumption of nonfat milk, but not whole milk, may result in a less atherogenic lipoprotein profile, and that the constituents of nonfat milk may improve lipid metabolism.

[Reversed clinicopathological conference (R-CPC)--interpreting laboratory data in the same way as physical findings].

Routine laboratory data are discussed by time series analysis in reversed clinicopathological conferences (R-CPC) at Shinshu University School of Medicine. We can identify fine changes in the laboratory data and the importance of negative data (without any changes) using time series analysis. Routine laboratory tests can be performed repeatedly and relatively cheaply, and time series analysis can be performed. The examination process of routine laboratory data in the R-CPC is almost the same as the process of taking physical findings. Firstly, general findings are checked and then the state of each organ is examined. Although routine laboratory data are cheap, we can obtain much more information about a patient's state than from physical examinations. In this R-CPC, several specialists in the various fields of laboratory medicine discussed the routine laboratory data of a patient, and we tried to understand the detailed state of the patient. R-CPC is an educational method to examine laboratory data and we, reconfirmed the usefulness of R-CPC to elucidate the clinical state of the patient.

[Case with intrauterine fetus death: interphase fluorescence in situ hybridization using buccal cells is useful for examining chromosomal abnormalities when placental villus not available].

The proband was a male fetus who died at 18 weeks of gestation. The fetus had growth retardation, hydrocephalus, exophthalmos, and micrognathia. The placental villus was not available. We performed interphase fluorescence in situ hybridization (FISH) using buccal cells of the fetus. The FISH using centromere specific probes for chromosome 7, 8 and 18, and RB1 gene (13q14)-specific probe showed three signals for each chromosome. The sex chromosome composition was XXY by FISH using centromere-specific probes for X and Y chromosomes. Thus, the fetus was diagnosed with triploidy syndrome. This report suggested that interphase FISH using buccal cells is useful for examining chromosomal abnormalities in intrauterine fetal death when placental villus is not available.

Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry.

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high-throughput methods, including ELISA.