High resolution melting curve assay for rapid detection of drug-resistant Mycobacterium tuberculosis.

We developed and evaluated a high resolution melting (HRM) curve assay by using real-time PCR for the detection of the most frequent mutations of Mycobacterium tuberculosis, which are responsible for the resistance of four anti-TB drugs: rifampicin, isoniazid, ethambutol, and streptomycin. The HRM assay was successfully used for the detection of dominant mutations: A516V, H526A, H526T, S531L, L533P, and A516G/S531L in rpoB; S315T, and S315A in katG; -15C/T, and -8T/C in mab-inhA; M306I in embB; K88Q and K43R in rpsL; and 513A/C in rrs. We were able to discriminate the mutant from the wild type by analyzing the melting-curve shape in 40 clinical M. tuberculosis isolates, and the results of the HRM assay were completely consistent with those of DNA sequencing. This HRM assay is a simple, rapid, and cost-effective method that can be performed in a closed tube. Therefore, our assay is a potentially useful tool for the rapid detection of drug-resistant M. tuberculosis.

Capture molecules preconditioned for kinetic analysis of high-affinity antigen-antibody complex in Biacore A100.

Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.

Soluble elastin decreases in the progress of atheroma formation in human aorta.

BACKGROUND: The serum levels of soluble elastin increase in patients with aortic dissection, but its distribution and characteristics are unclear. METHODS AND RESULTS: The 173 aortic specimens were categorized into 4 groups under microscopy (non-atherosclerotic aorta, n=13; fiber-rich plaque, n=77; lipid-rich plaque, n=66; ruptured plaque, n=17). Soluble elastin was abundant within the intima of both the non-atherosclerotic aorta and fiber-rich plaque, rather than in the media, and was decreased within the intima of lipid-rich and ruptured plaques. Soluble elastin levels decreased with progress of atherosclerosis (6.0 +/-0.3 microg/mg protein in non-atherosclerotic aorta; 5.8 +/-0.2 microg/mg protein in fiber-rich plaque; 4.9 +/-0.2 microg/mg protein in lipid-rich plaque; 2.8 +/-0.4 microg/mg protein in ruptured plaque, P<0.05). As well, both matrix metalloprotease-9 activity and elastin mRNA expression showed inverse distribution against soluble elastin (r=0.437, P<0.0001; r=0.186, P<0.05, respectively). Multivariable analysis revealed a decrease in the level of soluble elastin in ruptured plaque (2.8 +/-0.4 microg/mg protein in ruptured plaque, n=18; 5.5 +/-0.2 microg/mg protein in non-ruptured plaque, n=155, P<0.01). Furthermore, western blot showed soluble elastin consists of heterogeneous molecular pattern proteins. CONCLUSIONS: Both the synthesis and degradation of elastin may be enhanced in active atherosclerotic lesions.

Laminin gamma2-chain fragment in the circulation: a prognostic indicator of epithelial tumor invasion.

Laminin (LN) 5, the major component of epithelial-derived extracellular matrix (ECM), plays a major role in cell adhesion and motility. Previous reports stated that proteolytic processing of the NH(2)-terminal region of the gamma2 chain enhances cell motility on LN5, indicating that the degraded gamma2 chain NH(2)-terminal region would be shed from the ECM. However, soluble LN gamma2 NH(2)-terminal fragment (G2F) have not been detected in biological fluids. Here, we developed a double-monoclonal electrochemiluminescence immunoassay for human G2F and detected its presence in the normal human circulation (mean +/- SD: 39.2 +/- 10.3 ng/ml; n = 10). We also measured serum levels of G2F in nude mice orthotopically transplanted with three different human pancreatic carcinoma cell lines: MIApaca-II (secreting no LN5), HPAC (secreting the alpha3beta3gamma2 heterotrimer of LN5), or KP-1 (secreting the monomeric gamma2 chain of LN5). Serum levels of G2F drastically increased in the nude mice transplanted with HPAC (mean +/- SD: 351 +/- 33 ng/ml, 5 weeks after transplantation), the most invasive tumor cells to generate extensive peritoneal dissemination in vivo. A moderate increase in serum levels of G2F was also observed in mice transplanted with KP-1 (87.9 +/- 82 ng/ml, 5 weeks after transplantation), but no antigen was detected in the sera of MIApaca-II-transplanted mice. Therefore, circulating G2F was demonstrated to be a sensitive marker for LN5 production of primary pancreatic carcinomas, even if it was produced only as a monomeric gamma2 chain. In 11 established human pancreatic tumor cell lines (6 of LN5-producing cells and 5 of nonproducing cells), LN5-secreting cells have significantly higher levels of cell surface expression of beta4 integrin than nonsecreting cells. Thus, LN5 secretion is accompanied by cell surface expression of alpha6beta4 integrin, participating in hemidesmosome reorganization to form invading edges of malignant epithelial carcinomas. These data reveal that the level of circulating G2F is a new, prognostic, tumor-characterizing marker for estimating the invasiveness and malignancy of epithelial carcinomas in cancer patients.

[Blood concentration of laminin gamma2 chain in patients with head and neck cancer].

Laminin gamma2 chain (LN gamma2), expressed in human cancer cells and correlated with cancer malignancy, is cleaved by proteases and secreted into circulation. We measured the blood concentration of LN gamma2 in patients with head and neck cancer by an immuno-fluorescence assay using monoclonal antibodies against human LN gamma2. The normal blood concen- showed normal LN gamma2 concentration less than 50 ng/ml and 20 (33%) increased concentration exceeding 50 ng/ml. The relative ratio of the number of patients who showed increased LN gamma2 concentration correlated with the clinical stages of cancer. The blood concentration of LN gamma2 in 24 who initially showed normal concentrations did not change after radical treatments. Five who initially showed increased LN gamma2 concentration showed decreased concentration of less than 50 ng/ml after radical treatment. Four showed increased LN gamma2 concentration after treatment, and presented residual cancer, which killed them. Three of the 4 patients showed marked increase in LN gamma2 concentration exceeding 100 ng/ml and developed multiple distant metastases to the lung, liver, bone, and skin. The blood concentration of LN gamma2 in patients with head and neck cancer may thus indicate the amount of highly malignant cancer cells expressing LN gamma2. The blood concentration of LN gamma2 could therefore serve as a new tumor marker that might indicate the malignancy of and efficacy of treatments for head and neck cancer.

Usefulness of Measuring the Serum Elastin Fragment Level in the Diagnosis of an Acute Aortic Dissection.

Previous reports have shown that serum elastin fragments (SEFs) may be a useful biomarker for the diagnosis of an acute aortic dissection (AAD). However, because the reference interval of SEFs has not been established, it has not been determined whether SEFs are really useful for the diagnosis of AAD. The purpose of this study was to determine the usefulness of measuring SEFs for the diagnosis of AAD. A total of 42 consecutive patients aged 68 ± 18 years who were diagnosed with an AAD were studied. Patient background and SEF levels were examined on admission. SEF levels were also measured in patients undergoing a medical examination (n = 531, age 54 ± 17 years) to compare with those with an AAD. In the control group, SEF levels increased with age (R = 0.725, p <0.001). Then, we defined the upper limit of the reference interval of SEF levels as the 97.5th percentile of control SEF grouped by decade of life from the sixth to ninth decade. The overall risk of AAD exceeding the upper limit of the reference interval at each decade was 10% (4 of 42). For patients in their 60s and 70s, median SEF levels in the AAD group (89 [77 to 104], 93 [60 to 123] ng/ml, respectively) were not significantly higher than those in the control group (79 [68 to 92], 90 [79 to 106] ng/ml, respectively; p = 0.081 and 0.990, respectively). Our data suggest that measuring SEF levels may not be useful in the diagnosis of an AAD as the upper limit of the reference interval of the SEF level was unexpectedly higher.

Laminin gamma2-chain fragment circulating level increases in patients with metastatic pancreatic ductal cell adenocarcinomas.

Laminin-5 (LN5) is expressed solely by epithelial cells and considered to enhance cell migration after being cleaved by proteases, leading to the shedding of the N-terminal fragment of the LN5 gamma2-chain (G2F). We estimated circulating G2F level in 181 patients with various digestive diseases and 15 healthy subjects. The G2F level in pancreatic ductal cell adenocarcinoma patients with liver metastases was markedly elevated, but that in patients without liver metastases was not significantly elevated. The G2F levels in patients with benign pancreatic tumours (pancreatic cysts and intraductal papillary mucinous tumours) were similar to that in healthy volunteers. On the other hand, the level of the gamma1-chain is a common constituent of several laminin heterotrimers. The N-terminal fragment of gamma1-chain (G1F) in the circulation of these patients was also determined, and a slight increase in G1F levels was observed only in hepatocellular carcinoma patients. Interestingly, a significant increase in circulating G2F/G1F ratio was observed in patients with bile duct and gallbladder carcinoma, as well as in those with metastatic pancreatic ductal cell adenocarcinoma. The increase in the circulating G2F level, particularly compared with circulating G1F level, should correlated with LN5 overexpression in invasive carcinomas, independently of basement membrane metabolism in the entire body.

Laminin-5 in epithelial tumour invasion.

Laminin-5 (LN-5), consisting of alpha3-, beta3-, and gamma2-chains, is a component of the cell adhesion complex containing hemidesmosomes and anchoring fibrils. This protein is a major constituent of the extracellular matrix and has recently proved to be an invasion marker for epithelial cells in many immunohistochemical surveys, indicating that it is frequently expressed in the invading edges of epithelial tumour cells. Additionally, intracellular accumulation of monomeric gamma2-chains has been widely observed in the invasive carcinoma cells, but its mechanism was not entirely understood. Epithelial carcinoma cells prefer to adhere onto the LN-5-rich basement membranes using the specific integrins as receptors. Induction of cell migration is an important function of LN-5 and the enhanced activity is observed in its truncated form after proteolytic shedding of the N-terminal fragments of gamma2-chains. This processing was demonstrated to be mediated mainly by several kinds of matrix metalloproteinases. The degraded fragments of gamma2-chains, released from invading carcinomas, can be immunodetected in biological fluids and potentially utilized in the clinical diagnosis of various epithelial cancers. Here, we summarize the previous clinical investigations of LN-5 in epithelial tumour progression, and also discuss what it can regulate in the cell physiological events.

Age-adjusted level of circulating elastin as a cardiovascular risk factor in medical check-up individuals.

AIMS: The level of circulating soluble elastin (CSE) is reported to increase proportionally with the degree of clinical atherosclerosis; however, its diagnostic use is limited because CSE also increases with age. We aimed to investigate whether alterations in CSE concentrations are implicated in potential cardiovascular dysfunctions (indicated by standard physiological parameters) in medical check-up individuals, taking age into consideration. METHODS: In a total of 531 individuals (age 20-89 years), CSE levels were correlated most significantly with age. The groups of male and female individuals were each further divided into two subgroups: those with higher and those with lower CSE levels than the reference values determined by polynomial regression. RESULTS: Male participants with lower CSE levels (n = 128) than the age-adjusted reference baseline levels showed higher serum glucose (P < 0.008), uric acid (P < 0.008) and triglyceride (P < 0.02) levels than those with higher CSE levels (n = 126). However, most of the parameters tested in female participants with lower CSE levels (n = 140) were statistically comparable to those with higher CSE levels (n = 137). The ratio of CSE level to the age-adjusted reference level was calculated in each of the male participants, and declines in the ratio were significantly correlated with increases of serum glucose, uric acid and triglyceride levels (P < 0.005, P < 0.02 and P < 0.006, respectively). CONCLUSION: The decrease in age-adjusted CSE levels is a potential indicator of eventual cardiovascular dysfunction in medical check-up individuals, as predicted by the risk factors dyslipidemia, hyperuricemia or diabetes.

Phylogenetic analysis and seroprevalence of influenza C virus in Mie Prefecture, Japan in 2012.

Reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR were used to detect 14 (6.6%) influenza C virus (InfC) among 213 clinical samples collected from children with respiratory symptoms in Mie Prefecture, Japan, between January 2012 and December 2012. Virus isolation using Madin-Darby canine kidney cells and/or embryonated chicken eggs was also successful for 3 of the 14 PCR-positive samples. Eleven patients (78.6%) were aged <3 years. Phylogenetic analysis of the hemagglutinin-esterase gene showed that the InfC detected in Mie Prefecture belonged to the C/Sao Paulo/82-related lineage. To determine the seroprevalence of InfC, a total of 575 serum samples from patients aged 1 month to 69 years in Mie Prefecture were screened by hemagglutination inhibition test using the C/Mie/199/2012 (C/Sao Paulo/82-related lineage) strain as the antigen. The samples with an antibody titer of ≥1:16 were designated as antibody-positive. The results showed that 53.7% of the 296 serum samples collected in 2011 and 85.3% of the 279 samples collected in 2012 were positive for antibodies against InfC, suggesting that an outbreak of InfC infection occurred in Mie Prefecture in 2012. Therefore, continuous and proactive monitoring is important to determine the number of InfC-infections and to better understand the epidemiology.

Molecular genotyping of Mycobacterium tuberculosis in Mie Prefecture, Japan, using variable numbers of tandem repeats analysis.

The variable numbers of tandem repeats (VNTR) analysis is a method frequently employed as a molecular epidemiological tool for Mycobacterium tuberculosis genetic fingerprinting. In this study, we characterized the population of M. tuberculosis circulating in Mie Prefecture, Japan, and assessed the utility of the proposed JATA12- and 15-VNTR analyses of 158 M. tuberculosis clinical isolates using 25 VNTR loci. The results revealed that the ancient Beijing sublineage is the most prevalent M. tuberculosis strain in Mie Prefecture, accounting for 85.0% of 113 Beijing lineage isolates. Our results also showed that JATA-VNTR using well-selected loci is as reliable as standardized 15-locus MIRU-VNTR. Furthermore, JATA15-VNTR analysis reliably improved the discriminatory power compared with basic JATA12-VNTR analysis. In summary, our data suggest that JATA-VNTR is a useful tool for discrimination of M. tuberculosis in areas where ancient Beijing strains are frequently isolated.

Involvement of E-cadherin cleavage in reperfusion injury.

OBJECTIVE: E-cadherin is a major cell-to-cell adhesion molecule, of which the ectodomain is cleaved from epithelial cells to yield a soluble form after the pathological alteration of the alveolar epithelium. We investigated the excretion level of soluble E-cadherin in a rat lung isotransplant model, and demonstrated the involvement of this molecule in the pathogenesis of reperfusion injury after lung transplantation. METHODS: Inbred male Lewis rats were used as both donor and recipient animals, and they were subjected to left lung isotransplantation. After 6 h of ischaemia, the left lung was transplanted into a recipient rat and reperfused for 4 h. The animals were injected intravenously with (125)I-labelled albumin at 3 h after the onset of reperfusion as a marker of pulmonary albumin leakage. We assessed pulmonary alveolar septal damage quantitatively based on the (125)I-albumin concentration ratio of bronchoalveolar lavage fluid (BALF) to plasma. Soluble E-cadherin fragments were detected in BALF on Western blot analysis using affinity-purified antibodies specific to rat E-cadherin synthetic peptides. RESULTS: The BALF supernatant-to-plasma ratio of the graft lung was significantly increased compared to that of the control group. Western blot analysis showed a marked release of soluble E-cadherin into BALF, and its increase in BALF was associated with alveolar septal damage. CONCLUSIONS: These results suggest that one potential mechanism of lung reperfusion injury involves the cleavage of E-cadherin.

Laminin gamma2 fragments are increased in the circulation of patients with early phase acute lung injury.

OBJECTIVE: Laminin-5, a cell adhesive molecule expressed solely by epithelium, is known to enhance epithelial cell migration and repair of injured epithelium, after its essential component gamma2-chain is processed proteolytically. Our previous study revealed circulating levels of amino-terminal fragment of laminin gamma2-chain (G2F) reflect epithelial tumor invasiveness in carcinoma patients, but its physiological role in alveolar epithelial injury remains unknown. DESIGN: Sampling of epithelial lining fluids or pulmonary edema fluids from patients with acute lung injury (ALI) or related diseases was performed. Plasma samples were obtained from them at the time of disease onset or later. G2F concentrations were determined by immunoassay constructed by ourselves. RESULTS: We found a significantly higher amount of G2F in pulmonary edema and epithelial lining fluids of patients with ALI, as compared with those with the other respiratory diseases. Their plasma levels were also elevated significantly early at the onset of ALI (mean +/- SD; 147 +/- 82 ng/ml in non-surviving and 90 +/- 56 in surviving patients) as compared with those in the patients with cardiogenic pulmonary edema (59 +/- 36) or idiopathic pulmonary fibrosis (37 +/- 17), indicating alveolar epithelium rapidly secrete laminin-5 in ALI. At 5 days after onset, non-surviving patients maintained higher plasma concentrations (152 +/- 84), but in contrast, the levels in surviving patients declined (71 +/- 35), suggesting secretion of laminin-5 was suppressed, associated with recovery from ALI. CONCLUSION: Circulating G2F may be a biomarker for alveolar laminin-5 secreted early at disease onset in ALI, potentially regulating alveolar re-epithelialization.

Serum concentrations of laminin gamma2 fragments in patients with head and neck squamous cell carcinoma.

BACKGROUND: The laminin (LN) gamma2 chain expression has been linked to tumor invasion and prognosis. To provide a convenient clinical use, procedures that analyze LNgamma2 expression by using the serum and/or urine of patients should be developed. METHODS: The serum concentrations of the N-terminal fragments of the LNgamma2 chain in 73 patients with head and neck squamous cell carcinomas were measured by immunoassay. RESULTS: The concentrations of the LNgamma2 fragments ranged between 14.5 and 324.2 ng/mL, and the normal upper limit was estimated to be 50 ng/mL. The LNgamma2 fragment concentrations increased according to the T classification. The amount of elevated LNgamma2 fragment concentrations decreased after the use of curative treatments. Three patients displayed a continuous increase of the concentrations and subsequently died of the diseases. CONCLUSIONS: The serum concentrations of the LNgamma2 fragments may prove useful in assessing the treatment results and clinical courses of patients with head and neck squamous cell carcinomas.

Quantitative analysis for soluble elastin in circulation and cell culture fluids using monoclonal antibody-based sandwich immunoassay.

We have newly established 3 distinct murine monoclonal antibodies (MoAbs) against human soluble elastin by using chemically denatured immunogen isolated from human aorta; they are designated as HASG-2, HASG-30, and HASG-61-1. All of these MoAbs were highly reactive with soluble forms of native elastin in normal human serum. HASG-2 and HASG-61-1 MoAbs can recognize soluble bovine elastin as well as human antigen, but HASG-30 cannot. The sandwich enzyme-linked immunosorbent assay (ELISA) for human soluble elastin was developed with HASG-61-1 labeled with peroxidase and HASG-30 immobilized on the microplates. The circulating levels of soluble elastin in human healthy subjects (mean +/- SD; 42.9 +/- 19.9ng/mL; n = 85) could be measured with full accuracy and reproducibility, and gradually increased with aging. The positive correlation between the levels and ages was statistically significant (r = 0.581, p < 0.0001). In addition, we could also determine the concentration of tropoelastin secreted from cultured human dermal fibroblasts accurately by this ELISA. This simple assay can be utilized for the routine clinical laboratory screening of patients with arteriosclerotic vascular diseases or to accurately determine the concentrations of tropoelastin secreted from cultured human cells.