Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction.

The tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B12-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO2 by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.

Telbivudine Reduces Parvovirus B19-Induced Apoptosis in Circulating Angiogenic Cells.

Aims: Human parvovirus B19 (B19V) infection directly induces apoptosis and modulates CXCR4 expression of infected marrow-derived circulating angiogenic cells (CACs). This leads to dysfunctional endogenous vascular repair. Treatment for B19V-associated disease is restricted to symptomatic treatment. Telbivudine, a thymidine analogue, established in antiviral treatment for chronic hepatitis B, modulates pathways that might influence induction of apoptosis. Therefore, we tested the hypothesis of whether telbivudine influences B19V-induced apoptosis of CAC. Methods and Results: Pretreatment of two CAC-lines, early outgrowth endothelial progenitor cells (eo-EPC) and endothelial colony-forming cells (ECFC) with telbivudine before in vitro infection with B19V significantly reduced active caspase-3 protein expression (-39% and -40%, both p < 0.005). Expression of Baculoviral Inhibitor of apoptosis Repeat-Containing protein 3 (BIRC3) was significantly downregulated by in vitro B19V infection in ECFC measured by qRT-PCR. BIRC3 downregulation was abrogated with telbivudine pretreatment (p < 0.001). This was confirmed by single gene PCR (p = 0.017) and Western blot analysis. In contrast, the missing effect of B19V on angiogenic gene expression postulates a post-transcriptional modulation of CXCR4. Conclusions: We for the first time show a treatment approach to reduce B19V-induced apoptosis. Telbivudine reverses B19V-induced dysregulation of BIRC3, thus, intervening in the apoptosis pathway and protecting susceptible cells from cell death. This approach could lead to an effective B19V treatment to reduce B19V-related disease.

CCR5del32 genotype in human enteroviral cardiomyopathy leads to spontaneous virus clearance and improved outcome compared to wildtype CCR5.

BACKGROUND: Enteroviral cardiomyopathy is a life-threatening disease, and detection of enterovirus (EV) RNA in the initial endomyocardial biopsy is associated with adverse prognosis and increased mortality. Some patients with EV infection may spontaneously eliminate the virus and recover, whereas those with virus persistence deteriorate and progress to heart failure. Interferon-beta (IFN-ß) therapy eliminates the virus, resulting in increased survival of treated patients. CCR5 is expressed on antigen-presenting cells (both macrophages and dendritic cells) and immune effector cells (T-lymphocytes with memory/effector phenotype and natural killer cells). Its 32-bp deletion (CCR5del32) is the most frequent human coding sequence mutation. This study addresses the correlation of CCR5 polymorphism to the clinical course of EV infection and the necessity for IFN-ß treatment. METHODS: We examined 97 consecutive patients with chronic/inflammatory cardiomyopathy and biopsy-proven EV infection and reliable information on clinical outcomes by CCr5 genotyping. These data were evaluated in relation to virus persistence in follow-up biopsies and survival rates over a 15-year period. RESULTS: Genotyping revealed a strong correlation between the CCR5del32 genotype and spontaneous virus clearance with improved outcomes. All patients with CCR5del32 eliminated EV spontaneously and none of them died within the observed period. In the group of untreated CCR5 wildtype patients, 33% died (Kaplan-Meier log-rank p = 0.010). However, CCR5 wildtype individuals treated with IFN-ß are more likely to survive than without therapy (Kaplan-Meier log-rank p = 0.004) in identical proportions to individuals with the CCR5del32 genotype. CONCLUSIONS: These data suggest that CCR5 genotyping is a novel predictive genetic marker for the clinical course of human EV cardiomyopathies. Hereby clinicians can identify those EV positive individuals who will eliminate the virus spontaneously based on CCR5 phenotype and those patients with CCR5 wildtype genotype who would be eligible for immediate antiviral IFN-ß treatment to minimize irreversible cardiac damage.

NOD2- and disease-specific gene expression profiles of peripheral blood mononuclear cells from Crohn's disease patients.

AIM: To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells (PBMCs) from Crohn's disease (CD) patients in clinical remission. METHODS: Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotide-binding oligomerization domain 2 (NOD2), and PBMCs from wild-type (WT)-NOD2 patients, patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis. The cells were cultured with vitamin D, peptidoglycan (PGN) and lipopolysaccharide (LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative real-time PCR. NOD2- and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model. RESULTS: Employing microarray assays, a total of 267 genes were identified that were significantly up- or downregulated in PBMCs of WT-NOD2 patients, compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN (P < 0.05; threshold: ≥ 2-fold change). For further analysis by real-time PCR, genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria. In a larger cohort of patients and controls, a disease-associated expression pattern, with higher transcript levels in vitamin D-treated PBMCs from patients, was observed for three of these genes, CLEC5A (P < 0.030), lysozyme (LYZ; P < 0.047) and TREM1 (P < 0.023). Six genes were found to be expressed in a NOD2-dependent manner (CD101, P < 0.002; CLEC5A, P < 0.020; CXCL5, P < 0.009; IL-24, P < 0.044; ITGB2, P < 0.041; LYZ, P < 0.042). Interestingly, the highest transcript levels were observed in patients with heterozygous NOD2 mutations. CONCLUSION: Our data identify CLEC5A and LYZ as CD- and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes.

The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B12-dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564-4572, 2015, https://doi.org/10.1128/AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter-1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g-1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1. IMPORTANCE: In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.

Absent MicroRNAs in Different Tissues of Patients with Acquired Cardiomyopathy.

MicroRNAs (miRNAs) can be found in a wide range of tissues and body fluids, and their specific signatures can be used to determine diseases or predict clinical courses. The miRNA profiles in biological samples (tissue, serum, peripheral blood mononuclear cells or other body fluids) differ significantly even in the same patient and therefore have their own specificity for the presented condition. Complex profiles of deregulated miRNAs are of high interest, whereas the importance of non-expressed miRNAs was ignored. Since miRNAs regulate gene expression rather negatively, absent miRNAs could indicate genes with unaltered expression that therefore are normally expressed in specific compartments or under specific disease situations. For the first time, non-detectable miRNAs in different tissues and body fluids from patients with different diseases (cardiomyopathies, Alzheimer's disease, bladder cancer, and ocular cancer) were analyzed and compared in this study. miRNA expression data were generated by microarray or TaqMan PCR-based platforms. Lists of absent miRNAs of primarily cardiac patients (myocardium, blood cells, and serum) were clustered and analyzed for potentially involved pathways using two prediction platforms, i.e., miRNA enrichment analysis and annotation tool (miEAA) and DIANA miRPath. Extensive search in biomedical publication databases for the relevance of non-expressed miRNAs in predicted pathways revealed no evidence for their involvement in heart-related pathways as indicated by software tools, confirming proposed approach.

Calorespirometric feeding control enhances bioproduction from toxic feedstocks-Demonstration for biopolymer production out of methanol.

The sustainable production of fuels and industrial bulk chemicals by microorganisms in biotechnological processes is promising but still facing various challenges. In particular, toxic substrates require an efficient process control strategy. Methanol, as an example, has the potential to become a major future feedstock due to its availability from fossil and renewable resources. However, besides being toxic, methanol is highly volatile. To optimize its dosage during microbial cultivations, an innovative, predictive process control strategy based on calorespirometry, i.e., simultaneous measurements of heat and CO2 emission rates, was developed. This rarely used technique allows an online-estimation of growth parameters such as the specific growth rate and substrate consumption rate as well as a detection of shifts in microbial metabolism thus enabling an adapted feeding for different phases of growth. The calorespirometric control strategy is demonstrated exemplarily for growth of the methylotrophic bacterium Methylobacterium extorquens on methanol and compared to alternative control strategies. Applying the new approach, the methanol concentration could be maintained far below a critical limit, while increased growth rates of M. extorquens and higher final contents of the biopolymer polyhydroxybutyrate were obtained. Biotechnol. Bioeng. 2016;113: 2113-2121. © 2016 Wiley Periodicals, Inc.

Differential Cardiac MicroRNA Expression Predicts the Clinical Course in Human Enterovirus Cardiomyopathy.

BACKGROUND: Investigation of disease pathogenesis confined to protein-coding regions of the genome may be incomplete because many noncoding variants are associated with disease. We aimed to identify novel predictive markers for the course of enterovirus (CVB3) cardiomyopathy by screening for noncoding elements influencing the grossly different antiviral capacity of individual patients. METHODS AND RESULTS: Transcriptome mapping of CVB3 cardiomyopathy patients revealed distinctive cardiac microRNA (miR) patterns associated with spontaneous virus clearance and recovery (CVB3-ELIM) versus virus persistence and progressive clinical deterioration (CVB3-PERS). Profiling of protein-coding genes and 754 miRs in endomyocardial biopsies of test cohorts was performed at their initial presentation, and those spontaneously eliminating the virus were compared with those with virus persistence on follow-up. miR profiling revealed highly significant differences in cardiac levels of 16 miRs, but not of protein-coding genes. Evaluation of this primary distinctive miR pattern in validation cohorts, and multivariate receiver operating characteristic curve analysis, confirmed this pattern as highly predictive for disease course (area under the curve, 0.897±0.071; 95% confidence interval, 0.758-1.000). Eight miRs were strongly induced in CVB3-PERS (miRs 135b, 155, 190, 422a, 489, 590, 601, 1290), but undetectable in CVB3-ELIM or controls. They are predicted to target multiple immune response genes, and 2 of these were confirmed by antisense-mediated ablation of miRs 135b, 190, and 422a in the monocytic THP-1 cell line. CONCLUSIONS: An immediate clinical application of the data is cardiac miR profiling to assess the risk of virus persistence and progressive clinical deterioration in CVB3 cardiomyopathy. Patients at risk are eligible for immediate antiviral therapy to minimize irreversible cardiac damage.

Detection of clonal T-cell-receptor (TCR) Vbeta rearrangements in explanted dilated cardiomyopathy hearts by semi-nested PCR, GeneScan, and direct sequencing.

BACKGROUND: Viral infection and anti-cardiac immunity are involved in the pathogenesis of dilated cardiomyopathy (DCM). Immunity targeting particular antigens may evoke expansion of reactive T-cell clones. MATERIAL AND METHODS: Myocardial tissues from explanted hearts were investigated for clonal T-cell-receptor- (TCR-) ß rearrangements by an established semi-nested polymerase chain reaction (PCR), followed by high-resolution GeneScan analysis and direct sequencing. From 17 explanted DCM hearts, 3 myocardial samples each were obtained from the right ventricle, the septum, and the left ventricle (total: 9 myocardial samples per case). Six explanted hearts with non-DCM cardiomyopathy entities served as controls. RESULTS: GeneScan analysis revealed polyclonal TCR-ß rearrangements in all controls. In contrast, at least 1 myocardial sample in 9 out of 17 DCM hearts (total: 20 of the 81 DCM specimens) displayed single dominant TCR-ß PCR products consistent with the presence of clonal T-cell populations. Direct sequencing of the clonal TCR-ß PCR-products disclosed an involvement of Vß 19.01 segments in 14 of the dominant amplificates (70%). Further TCR-Vß segments involved in clonal TCR-ß rearrangements of DCM hearts were Vß 6-1.01 (n=1), Vß 6-3.01 (n=2), Vß 6-5.01 (n=1), Vß 10-3.02 (n=1), and Vß 19.03 (n=1). CONCLUSIONS: The detectability of clonal TCR-ß rearrangements indicates a pathogenic relevance of this finding in DCM. The predominance of Vß 19.01 segments suggests that the immune response in DCM patients targets particular epitopes. However, the partly heterogenic TCR-ß populations in various myocardial samples from the respective cases support the notion that T-cell immunity may target multiple epitopes in human DCM.

Selective regulation of cardiac organic cation transporter novel type 2 (OCTN2) in dilated cardiomyopathy.

Organic cation transporters (OCT1-3 and OCTN1/2) facilitate cardiac uptake of endogenous compounds and numerous drugs. Genetic variants of OCTN2, for example, reduce uptake of carnitine, leading to heart failure. Whether expression and function of OCTs and OCTNs are altered by disease has not been explored in detail. We therefore studied cardiac expression, heart failure-dependent regulation, and affinity to cardiovascular drugs of these transporters. Cardiac transporter mRNA levels were OCTN2>OCT3>OCTN1>OCT1 (OCT2 was not detected). Proteins were localized in vascular structures (OCT3/OCTN2/OCTN1) and cardiomyocytes (OCT1/OCTN1). Functional studies revealed a specific drug-interaction profile with pronounced inhibition of OCT1 function, for example, carvedilol [half maximal inhibitory concentration (IC50), 1.4 µmol/L], diltiazem (IC50, 1.7 µmol/L), or propafenone (IC50, 1.0 µmol/L). With use of the cardiomyopathy model of coxsackievirus-infected mice, Octn2mRNA expression was significantly reduced (56% of controls, 8 days after infection). Accordingly, in endomyocardial biopsy specimens OCTN2 expression was significantly reduced in patients with dilated cardiomyopathy, whereas the expression of OCT1-3 and OCTN1 was not affected. For OCTN2 we observed a significant correlation between expression and left ventricular ejection fraction (r = 0.53, P < 0.0001) and the presence of cardiac CD3⁺ T cells (r = -0.45, P < 0.05), respectively. OCT1, OCT3, OCTN1, and OCTN2 are expressed in the human heart and interact with cardiovascular drugs. OCTN2 expression is selectively reduced in dilated cardiomyopathy patients and predicts the impairment of cardiac function.

Expression of functional T-cell markers and T-cell receptor Vbeta repertoire in endomyocardial biopsies from patients presenting with acute myocarditis and dilated cardiomyopathy.

AIMS: To quantify and functionally characterize the intramyocardial T-cells in endomyocardial biopsies (EMBs) from patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM). METHODS AND RESULTS: Expression of genes characterizing Th1 [interferon (IFN)γ, Tbet-1, Eomesodermin, interleukin (IL)-27], Th2 (IL-4, IL-5, GATA3), Th17 (IL-17), regulatory [regulatory T-cells (Treg); FoxP3, TGFß, IL-10], anergic (GRAIL), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A), as well as of functional T-cell receptor Vbeta (TRBV) families were investigated in EMBs from AMC patients (n= 58) and DCM patients (n= 34) by pre-amplified real-time reverse transcription-polymerase chain reaction. These data were compared with EMBs from n= 19 controls. Expression of CD3d, CD3z, and TRBC (T-cell receptor beta constant region) were associated with the immunohistological diagnosis of inflammatory cardiomyopathy (DCMi). In EMBs from DCM patients with increased CD3d expression, significantly increased markers of Th1 (IFNγ, T-bet, Eomesodermin), regulatory T-cells (Treg; FoxP3, TGFß), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A) were present, while no differential polarization of T-cells was found in EMBs form AMC patients. A differential dominance of distinct functional TRBV families was associated with different cardiotropic viruses: TRBV 11 and 24 with Parvovirus B19; TRBV4, 10 and 28 with human herpes virus type 6; and TRBV14 for Coxsackie virus, respectively. CONCLUSIONS: The T-cell infiltrates in human DCMi are characterized by differential expression of functional T-cell markers indicating Th1, Treg, and CTLs, while no major role could be confirmed for Th17. The virus-associated differential TRBV dominance suggests an antiviral specificity of virus-induced T-cell responses in human DCMi.

Generation of HCMV-specific T-cell lines from seropositive solid-organ-transplant recipients for adoptive T-cell therapy.

Chronically immunosuppressed patients, like solid-organ-transplant (SOT) recipients, are at increased risk for severe human cytomegalovirus (HCMV) infection. Despite the availability of effective antiviral drugs, lasting control of remaining viruses is dependent on an effective T-cell immunity. So in some patients conventional antiviral therapy cannot control the infection and prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Selective reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells derived from healthy donors is a safe and effective approach in hematopoietic stem cell transplant recipients. The aim of this study was to determine whether functional HCMV-specific T cells can also be generated from chronically immunosuppressed patients. Autologous CD4+/8+ T-cell lines directed against the HCMV protein IE-1 were generated from the peripheral blood of SOT patients using a recently developed modular protocol easily applicable to good-manufacturing-practice conditions. T-cell lines from SOT patients showed similar features as cells from healthy donors regarding phenotype, functionality (HCMV-specific killing, gene expression pattern, and cytokine secretion), IE-1 epitope recognition, and dominance of distinct T-cell receptor V beta families. Most importantly, this protocol also allowed the generation of T-cell lines from immunosuppressed patients with HCMV infection/chronic HCMV disease. Our data suggest the potential of this autologous approach for the treatment of SOT recipients suffering from HCMV infection/disease poorly responding to virostatic therapy.

NS1 specific CD8+ T-cells with effector function and TRBV11 dominance in a patient with parvovirus B19 associated inflammatory cardiomyopathy.

BACKGROUND: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. METHODOLOGY AND PRINCIPAL FINDINGS: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mug nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amino-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNgamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNgamma, IL2, IL27 and T-bet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. CONCLUSIONS: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity.

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

Impact of cell culture media on the expansion efficiency and T-cell receptor Vbeta (TRBV) repertoire of in vitro expanded T cells using feeder cells.

BACKGROUND: The effects of different cell culture media on expansion efficiency and alterations in T-cell receptor V beta (TRBV) expression of in vitro expanded lymphocytes are not well established. MATERIAL/METHODS: Low numbers of CD3+ T cells from peripheral blood lymphocytes of healthy donors were subjected to polyclonal in vitro expansion in the presence of autologous CD3-depleted mononuclear cells as feeder cells (FCs) and their numbers and TRBV expressions were compared in media containing human (HS-RPMI) or fetal bovine serum (FBS-RPMI), Panserin413, or X-Vivo 15TM designed for lymphocyte culture. RESULTS: During three courses of restimulation within 28 days with CD3-antibody (OKT-3), IL-2, and initial CD3+, T-cell: FC ratios of 1:50 lowered to 1:5 and T cells expanded more than 1,000-fold in the media containing complete sera. Loss of cluster formation, associated with expansion failure, was only observed in cultures using synthetic media and resulted in only about 70-fold expansion. Whereas TRVB expression as determined by real-time PCR was substantially altered after 14 days of culture in X-Vivo 15, at day 28 only T cells from long-term culture in HS-RPMI presented the initial TRBV composition. CONCLUSIONS: Culture media have substantial impact on in vitro T-cell expansion. In the presence of FCs, medium containing human serum is superior to synthetic media and FBS-RPMI for long-term culture regarding T-cell number and TRBV repertoire. In contrast, the synthetic media Panserin413 and XVivo15 show lower expansion efficiency and reproducibility and, as RPMI1640+10%FBS, can contribute to overstimulation of certain TRBVs at advanced culture time points.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies.

BACKGROUND: Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). RESULTS: T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 +/- 0.33 Ct values. The coefficients for inter- (1.89 +/- 0.48%) and intra-assay variation (0.85 +/- 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 +/- 0.33, without significant differences between self-designed and ABI inventoried Taqman(R) gene assays. Only two of the tested Taqman(R) ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 +/- 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 +/- 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 +/- 0.74), albeit higher inter-assay CVs (5.38 +/- 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts. CONCLUSION: In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.