Quantitative PCR Primers Research Articles

Bartonella clarridgeiaein Fleas, Tahiti, French Polynesia

To the Editor: Bartonella species are small, gram-negative, fastidious, and hemotropic emerging pathogens that cause various human diseases and circulate between a large variety of mammalian and arthropod vectors. More than 30 Bartonella species have been isolated from humans as well as from wild and domestic animals worldwide (1). B. clarridgeiae was suggested to be a minor causative agent of cat-scratch disease (CSD) in humans, however, this suggestion remains controversial. Usually, the agent of CSD is B. henselae and its principal reservoir is domestic cats (Felis catus) (1,2). The principal vector of these 2 species is the cat flea (Ctenocephalides felis) (3,4). We report Bartonella species in fleas collected from cats and dogs in Tahiti, French Polynesia. In October 2009, fleas were collected from 1 cat and 9 dogs in Papeete, capital of Tahiti Island, French Polynesia. Fleas collected were kept in 70% ethanol and sent to the military veterinary service in Marseille, France; these fleas were later sent to Unite de Recherche sur les Maladies Infectieuses et Tropicales Emergentes in Marseilles. The fleas were identified phenotypically by using current taxonomic criteria. DNA from fleas and negative control DNA from noninfected laboratory lice were extracted by using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany), as described (3). Flea samples were tested for Bartonella spp. DNA by using the 7900 HT Fast Quantitative Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and primers and Taqman probes specific for the 16S–23S rRNA gene intergenic spacer region as described (5). Fleas were considered positive when cycle threshold was <30. All positive fleas at screening were confirmed by using standard Bartonella PCR and sequencing of partial internal transcribed spacer gene fragments by using primers URBarto1 and URBarto2, as described (3). B. elizabethae DNA was used as positive control. DNA sequencing reagents were obtained with BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM; Applied Biosystems). The sequences were assembled in Sequencher 4.2 (GeneCodes 2003; www.genecodes.com) and were compared with Bartonella sequences available in GenBank. Overall, 81 fleas were collected from 1 cat (13 fleas) and 9 dogs (68 fleas). All 81 fleas collected were morphologically identified as C. felis. Sample fleas were collected from animals visiting a veterinary clinic for neutering or vaccinations. The overall rate of Bartonella-positive fleas by molecular screening with real-time PCR was 7.4% (6/81): 6 fleas from the cat (6/13) and none from a dog (0/68). These positive samples were confirmed after intergenic spacer PCR amplification and sequencing with sequences at 100% identity with B. clarridgeiae (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU589237,term_id:187251975,term_text:EU589237}}EU589237). B. clarridgeiae was first isolated from the pet cat of an HIV-positive patient in the United States (6). However, B. clarridgeiae has never been isolated or detected by molecular methods in humans, and thus its implication as a human pathogen remains controversial. The presence of B. clarridgeiae antibodies has been reported in a suspected case of CSD and in a patient with a chest-wall abscess (4). However, B. clarridgeiae has been detected on fleas from various continents, including Europe, Asia, North America (1), Africa; New Zealand, and recently from New Caledonia (7). In France, several studies have reported the molecular detection of B. clarridgeiae in the blood of a cat or in cat fleas (C. felis), indicating the potential role of fleas as vectors of this organism (8,9). Prevalence of this bacterium in cat fleas may vary and be as high as 67.9% in cat fleas from France (3). Moreover, DNA of B. henselae and B. clarridgeiae has been reported from cat fleas from New Zealand (10). Similarly, co-infection with B. clarridgeiae and B. henselae has been reported in domestic cats from Europe and Asia (1). In our study, all Bartonella spp.–positive fleas harbored B. clarridgeiae only; all were obtained from cats and none from dogs, similar to findings reported from New Zealand (10), although B. clarridgeiae has been reported from a flea on a dog in Taiwan (2). Papeete, the capital of Tahiti, is located in the South Pacific Ocean, and remains one of the most visited areas by tourists from all over the world. There are many stray cats and dogs in Tahiti that may be infected with Bartonella species and thus serve as a reservoir for these pathogens. Our result confirms the presence of B. clarridgeiae in Tahiti and is a warning of the presence of flea-borne bartonellosis and the potential risk of B. clarridgeiae or other flea-borne diseases for humans exposed to cat fleas.

Stable‐isotope probing of the polycyclic aromatic hydrocarbon‐degrading bacterial guild in a contaminated soil

The bacteria responsible for the degradation of naphthalene, phenanthrene, pyrene, fluoranthene or benz[a]anthracene in a polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated by DNA-based stable-isotope probing (SIP). Clone libraries of 16S rRNA genes were generated from the (13) C-enriched ('heavy') DNA recovered from each SIP experiment, and quantitative PCR primers targeting the 16S rRNA gene were developed to measure the abundances of many of the SIP-identified sequences. Clone libraries from the SIP experiments with naphthalene, phenanthrene and fluoranthene primarily contained sequences related to bacteria previously associated with the degradation of those compounds. However, Pigmentiphaga-related sequences were newly associated with naphthalene and phenanthrene degradation, and sequences from a group of uncultivated γ-Proteobacteria known as Pyrene Group 2 were newly associated with fluoranthene and benz[a]anthracene degradation. Pyrene Group 2-related sequences were the only sequences recovered from the clone library generated from SIP with pyrene, and they were 82% of the sequences recovered from the clone library generated from SIP with benz[a]anthracene. In time-course experiments with each substrate in unlabelled form, the abundance of each of the measured groups increased in response to the corresponding substrate. These results provide a comprehensive description of the microbial ecology of a PAH-contaminated soil as it relates to the biodegradation of PAHs from two to four rings, and they underscore that bacteria in Pyrene Group 2 are well-suited for the degradation of four-ring PAHs.

Development of Porphyromonas gingivalis-specific quantitative real-time PCR primers based on the nucleotide sequence of rpoB

Species-specific quantitative real-time PCR (qPCR) primers were developed for the detection of Porphyromonas gingivalis. These primers, Pg-F/Pg-R, were designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). Species-specific amplicons were obtained from the tested P. gingivalis strains but not in any of the other strains (46 strains of 46 species). The qPCR primers could detect as little as 4 fg of P. gingivalis chromosomal DNA. These findings suggest that these qPCR primers are suitable for applications in epidemiological studies.

Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E₂ (PGE₂) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE₂. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE₂ secretion. Thus, ACSL4 modulates PGE₂ release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall.

Blocking O-Linked GlcNAc Cycling in Drosophila Insulin-producing Cells Perturbs Glucose-Insulin Homeostasis

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.

Deletion of the Tumor Suppressor Gene NF1 Is Found In 3.5% of 485 De Novo Adult Myeloid Leukemia and Is Correlated with Unfavourable Cytogenetic: On Behalf of the ALFA Group

AbstractAbstract 4171NF1 acts as a tumor-suppressor gene by encoding neurofibromin1, a GTPase-activating protein (GAP) inhibiting Ras signaling pathway. Germline mutations or microdeletions of NF1 are responsible for neurofibromatosis type 1, and the somatic loss of the remained wild-type allele lead to malignant tumors or juvenile myelomonocytic leukemia (JMML). Furthermore, several studies revealed heterozygous somatic deletions of the 17q11.2 region including NF1 in adult myeloid malignancies. The reported frequencies of this abnormality varied between 2.6% and 11% in AML and this variation can be attributable to heterogeneity or size of the analysed cohorts.Previously, we analyzed 131 de novo AML cases (AML3 excluded) by Agilent™ 105K microarrays. 6/131 cases (4.6%) showed somatic deletions in 17q11.2, including a small minimal deleted region of 300 kb comprising the entire NF1 gene. To further investigate the incidence of NF1 deletion in de novo AML, 354 additional patients were therefore screened for the deletion by quantitative real-time PCR (Primers and TaqMan-based probe Hs 01778367_cn from Applied Biosystems), and FISH (NF1/MPO probe KBI-40144 from Kreatech) was performed to confirm the loss of NF1 copy number. Altogether, heterozygous NF1 deletion was observed in 17/485 (3.5%) de novo AML. Clinico-biological data were available from 14 NF1 deleted patients and 380 non-deleted patients included in the ALFA-9801 and 9802 French Trials. There were no significant differences between the 2 groups in age, sex ratio, leukocytosis, FAB classification of AML, mutational status of FLT3, NPM1, CEBPα and IDH. Interestingly, NF1 deletion was significantly correlated with unfavourable cytogenetic (50% vs 18%, p=0.008) and especially with monosomal karyotype (29% vs 9%, p=0.03). However, no statistical significant differences were observed for complete remission rate, relapse risk 3 years after diagnosis and 3-years overall survival. Screening for bi-allelic inactivation by sequencing the remained allele in NF1 deleted patients is in progress.We next evaluated NF1 gene expression for 93 patients of our cohort (3 with NF1 deletion and 90 without) by Affymetrix U133 Plus 2.0 microarrays. The 3 NF1 deleted patients revealed a significant reduced mean of NF1 expression level. Interestingly, about 10% of the NF1 non-deleted patients presented a similar decrease in NF1 expression rate. This suggests that mechanisms for transcriptional regulation (such as mutations or epigenetic silencing of NF1) may also contribute to AML pathogenesis.In conclusion, NF1 deletions occur in only 3.5% of de novo AML and are associated with unfavourable cytogenetic. This relatively low frequency of NF1 deletion can however be counterbalanced by others alterations acting at the transcriptional level and this remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Evaluation of the systemic acute phase response and endometrial gene expression of serum amyloid A and pro- and anti-inflammatory cytokines in mares with experimentally induced endometritis

Infectious infertility in the mare is clinically well described, little is however known about the systemic acute phase reaction (APR) and local immunological responses accompanying equine endometritis. The aim of this study was to monitor selected markers of the APR in the systemic circulation and to correlate them to the local innate immune response in the uterus during infectious endometritis. Six adult standard bred mares received an intrauterine infusion of 10 9 CFU Escherichia coli. Blood samples were obtained before (0 h) and 3, 6, 12, 24, 36, 48, 72, 96 and 120 h post inoculation (pi), and endometrial biopsies were sampled before, and 3, 12, 24, 48 and 72 h pi. The infectious endometritis elicited a systemic APR with significantly increased concentrations of the acute phase proteins (APPs) serum amyloid A (SAA) and fibrinogen. Relative gene expression analyses were performed on extracted RNA from endometrial biopsies using quantitative real-time PCR and specific primers for SAA and pro- and anti-inflammatory cytokines. Expression of SAA was significantly up-regulated at 3 and 12 h pi, and a significant up-regulated expression of IL-1β, TNFα, IL-8 and IL-10 was observed at 3 h pi. Plasma concentration of SAA was significantly correlated to endometrial SAA expression. The results of the present study demonstrate that endometritis gives rise to a systemic APR and an up-regulated endometrial gene expression of SAA and several pro-and anti-inflammatory cytokines. Understanding endometrial expression of acute phase proteins and selected cytokines contributing to uterine immunity in equine endometritis could improve understanding of events leading to infertility in the mare and help identify candidate genes of mediators/markers for diagnostic use.

Evidence for NK cells in equine endometrial cups (43.29)

Abstract Placentation during equine pregnancy is distinguished by a unique subset of trophoblast cells that invade the endometrium and form discrete structures termed the endometrial cups. Shortly after the trophoblasts invade, there is a dramatic infiltration of maternal leukocytes which accumulate around the cups. Our lab has previously characterized the infiltrating leukocytes as primarily CD3+ cells with substantial CD4+ and CD8+ subpopulations. Here, we identify expression of the NK cell marker NKp46 in peripheral blood mononuclear cells (PBMC) and endometrial cup lymphocytes (ECL). PCR primers were developed to detect the equine homolog of NKp46 and expression was detected in resting and stimulated lymphocytes using standard PCR. Quantitative real-time PCR primers and standards were used to quantitate expression in CD3+ and CD3- sorted lymphocytes. NKp46 gene expression in CD3- lymphocytes was 10-fold higher than in CD3+ cells. Levels comparable to CD3-depleted lymphocytes were detected in frozen tissue sections of endometrial cups. The highest levels of expression were detected in leukocytes isolated from the endometrium surrounding the cups of five mares during early pregnancy (days 43-46). NKp46 expression levels were 7-43 fold higher in ECL compared to paired PBMC (p=0.04). Coincident CD3γ expression was lower in all ECL compared to paired PBMC (p=0.03). These data provide the first evidence for the presence of NK cells in the equine endometrium during pregnancy.

Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

PU.1 Directly Regulates cdk6 Gene Expression, Linking the Cell Proliferation and Differentiation Programs in Erythroid Cells

Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G(1) phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G(1) phase of the cell cycle, the period during which the decision to differentiate is made.

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium, all culture experiments to date have focused solely on representatives from one clade of Trichodesmium. Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB, idiA and feoB, we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB, we found that high isiB expression at low Fe levels corresponded to specific reductions in N(2) fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N(2) fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean.

Deep Sequencing of a Dimethylsulfoniopropionate-Degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data

In silico design and testing of environmental primer pairs with metagenomic data are beneficial for capturing a greater proportion of the natural sequence heterogeneity in microbial functional genes, as well as for understanding limitations of existing primer sets that were designed from more restricted sequence data. PCR primer pairs targeting 10 environmental clades and subclades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of thousands of dmdA sequences captured in marine metagenomic data sets. Using the bioinformatically optimized primers, dmdA genes were amplified from composite free-living coastal bacterioplankton DNA (from 38 samples over 5 years and two locations) and sequenced using 454 technology. An average of 6,400 amplicons per primer pair represented more than 700 clusters of environmental dmdA sequences across all primers, with clusters defined conservatively at >90% nucleotide sequence identity (approximately 95% amino acid identity). Degenerate and inosine-based primers did not perform better than specific primer pairs in determining dmdA richness and sometimes captured a lower degree of richness of sequences from the same DNA sample. A comparison of dmdA sequences in free-living versus particle-associated bacteria in southeastern U.S. coastal waters showed that sequence richness in some dmdA subgroups differed significantly between size fractions, though most gene clusters were shared (52 to 91%) and most sequences were affiliated with the shared clusters (approximately 90%). The availability of metagenomic sequence data has significantly enhanced the design of quantitative PCR primer pairs for this key functional gene, providing robust access to the capabilities and activities of DMSP demethylating bacteria in situ.

HHV-6 Reactivation Lowers the Risk of Acute Graft-Versus-Host Disease but Enhances the Risk of Infections Due to Activation of CD4+FoxP3+ Lymphocytes.

Abstract 4653CMV, EBV, and HHV-6 reactivation was monitored for 20 days to 4 years after allogeneic hematopoietic stem cell transplantation (alloHSCT) with respect to hematological and immunological reconstitution as well as fate in 118 patients (17 children and 101 adults) who received alloHSCT (100 peripheral blood progenitor cell (PBPC) and 18 bone marrow transplants from 53 siblings and 65 unrelated donors, 64 patients receiving reduced intensive conditioning (RIC) and 54 myeloablative conditioning). All patients on RIC and those transplanted from unrelated donors received anti-thymocyte globulin (ATG). Herpes virus DNA copies were measured by quantitative PCR (primers and probes designed according to Kimura 1999, Machida 2000, and Locatelli 2000). This study focused on HHV-6.It was found that:1.Multivariate analysis showed that alternative donor (OR=3.06, p=0.038), myeloablative conditioning regimen (OR=0.22, p=0.004), and an HLA-matched (10/10 at allele level) graft (OR=0.14, p=0.011) were risk factors of HHV-6 reactivation. Interestingly, they were different from those of CMV reactivation (acute GvHD grade >=I, OR=2.74, p=0.024).2.HHV-6 reactivation was found earlier post-transplant than CMV reactivation (19 vs. 47 days, median values, p=0.014) and rather before clinically apparent aGvHD manifestation, in contrast to CMV and EBV infections (percentages of reactivation events prior to aGvHD were 71, 14, and 14 for HHV-6, CMV and EBV, respectively).3.HHV-6 was associated with an increase in CD4+ lymphocytes (p=0.028, Wilcoxon matched pairs test), in contrast to CMV infection, in which CD4+ cells were rather low.4.HHV-6 reactivation noted during 100 days post-transplant was associated with a higher fraction of FoxP3+ in CD4+ cells compared with those of counterparts (13% vs. 8.5%, median values, p=0.008).5.Encephalitis was frequent in HHV-6 (8/14 vs. 26/104, p=0.023) and EBV (9/14 vs. 22/104, p=0.002) positive patients. HHV-6 preceded EBV (14/26 vs. 28/92, p=0.037) but not CMV (9/36 vs. 33/82, p=0.145) reactivation before 100 days after HSCT.6.HHV-6 positivity in the 100 days post-HSCT was associated with a higher mortality rate early post-transplant (6-month survival rates of HHV-6-positive and -negative cases: 55% vs. 80%, respectively).In conclusion: HHV-6 reactivation affects patients early post-transplant and associates with an increase in CD4+ lymphocytes in blood which frequently have FoxP3 generation potential. Risk of aGvHD is consequently lower, but there is a higher incidence of infections, including EBV, encephalitis, and sepsis, and survival is poor. Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Leukemia Antigen CML66 Elicits Coordinated B and T Cell Immunity After Donor Lymphocyte Infusion.

Abstract 2449Poster Board II-426Donor lymphocyte infusion (DLI) is a highly effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic hematopoietic stem cell transplantation (HSCT), in which 70-80% patients achieve curative responses. Despite its efficacy and established clinical use, the mechanism by which DLI achieves anti-tumor immunity remains incompletely understood. CML66 was discovered as an overexpressed nonpolymorphic CML-associated antigen that elicited a high-titer (>1:50,000) antibody response in a patient who received CD4+ DLI for relapsed CML. CML66 is preferentially expressed on myeloid leukemia progenitor cells and the development of CML66 antibodies temporally correlated with attainment of molecular remission. Since CD4+ T cell responses are involved in generating both antigen-specific B cell and CD8+T cell responses, we queried whether development of potent B cell immunity was also associated with the development of cytolytic T cell immunity against CML66. To detect specific T cell responses, post-DLI PBMC from this patient were screened for reactivity against pools of overlapping 15- to 18-mer peptides encoding the entire protein sequence of CML66. Patient CD8+ T cells were found to be reactive against a single CML66-derived peptide pool, and specifically an 8 amino acid peptide (HDVDALLW), beginning at residue 459. This T cell epitope is positioned in a different region of the protein compared to the B cell epitope (at residues 198-217: GFYVSLEWVTISKKNQDNK). A peptide-specific T cell clone was isolated, whose MHC class I molecule binding was restricted to HLA B*4403. The CML66-reactive T cell clone recognized the HLA-B4403-expressing CD34+ AML cell line MUTZ-3, providing evidence that this epitope is naturally processed and presented by myeloid leukemia cells. Functional cytotoxic T cell responses to CML66 appeared to precede the appearance of antigen-specific antibody responses. While CML66-specific plasma antibody was only detectable starting 2-3 months after DLI, reactivity to the HLA-B4403-restricted epitope by peripheral blood-derived CD8+ T cells was detected by IFNγ ELISpot as early as one month following DLI. To sensitively detect CD8+ CML66-specific T cells in vivo, we sequenced the CDR3 region specific to the CML66-specific clone, and designed clone-specific quantitative PCR primers. Nested PCR of RNA directly extracted from patient PBMC revealed only minimal detection of the CML66-specific T cell clone prior to DLI, but then expansion between 1 to 6 months after DLI. Similar analysis of marrow-derived RNA also revealed the presence of CML66-specific T cell clone in patient marrow following DLI. Moreover, this clone was already detected in patient marrow prior to DLI, suggesting that marrow is a reservoir of leukemia-specific T cells following hematopoietic stem cell transplantation. Our studies provide the first description of coordinated adaptive immunity developing against a nonpolymorphic leukemia-associated antigen, in close temporal correlation with the attainment of long-lasting immune-mediated clinical remission after allogeneic HSCT. The detected cytolytic T cell response was associated with the secretion of high-titer IgG antibody specific for a distinct epitope in the same target protein. In this setting, our results indicate that infusion of CD4+ donor cells resulted in the rapid activation and expansion of pre-existing marrow-resident leukemia-specific CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in the periphery. Disclosures:No relevant conflicts of interest to declare.

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.

Infection decreases fatty acid oxidation and nuclear hormone receptors in the diaphragm

Respiratory failure is a major cause of mortality during septic shock and is due in part to decreased ventilatory muscle contraction. Ventilatory muscles have high energy demands; fatty acid (FA) oxidation is an important source of ATP. FA oxidation is regulated by nuclear hormone receptors; studies have shown that the expression of these receptors is decreased in liver, heart, and kidney during sepsis. Here, we demonstrate that lipopolysaccharide (LPS) decreases FA oxidation and the expression of lipoprotein lipase (LPL), FA transport protein 1 (FATP-1), CD36, carnitine palmitoyltransferase beta, medium chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA synthetase, key proteins required for FA uptake and oxidation, in the diaphragm. LPS also decreased mRNA levels of PPARalpha and beta/delta, RXRalpha, beta, and gamma, thyroid hormone receptor alpha and beta, and estrogen related receptor alpha (ERRalpha) and their coactivators PGC-1alpha, PGC-1beta, SRC1, SRC2, Lipin 1, and CBP. Zymosan resulted in similar changes in the diaphragm. Finally, in PPARalpha deficient mice, baseline CPT-1beta and FATP-1 levels were markedly decreased and were not further reduced by LPS suggesting that a decrease in the PPARalpha signaling pathway plays an important role in inducing some of these changes. The decrease in FA oxidation in the diaphragm may be detrimental, leading to decreased diaphragm contraction and an increased risk of respiratory failure during sepsis.

Development of clade-specific Symbiodinium primers for quantitative PCR (qPCR) and their application to detecting clade D symbionts in Caribbean corals

We developed quantitative PCR (qPCR) assays to distinguish each of the four clades (A-D) of dinofla- gellate endosymbionts (genus Symbiodinium) commonly found in Caribbean corals. We applied these primer sets, which target portions of the multi-copy ribosomal DNA (rDNA) gene family, to assess the presence/absence of symbionts in clade D (as indicated by the detection of clade D DNA). We detected these symbionts in five of six Caribbean host species/genera (21% of samples analyzed, N = 10 of 47 colonies), from which clade D had rarely or never been observed. This suggests that Symbiodinium in clade D are present in a higher diversity of coral species than previously thought. This qPCR-based approach can improve our understanding of the total microbial diversity associated with corals, particularly in hosts thought to be relatively specific, and has many other potential applica- tions for studies of coral reef ecology and conservation.

Molecular Biological Detection and Quantification of Novel Fibrobacter Populations in Freshwater Lakes

PCR and quantitative PCR (qPCR) primers targeting the 16S rRNA gene were used to detect and quantify members of the genus Fibrobacter in lake water, sediment and colonized cotton taken from two freshwater lakes. Phylogenetic analysis identified two groups of sequences; those clustered with Fibrobacter succinogenes, the type species, and a defined cluster of clones loosely associated with several Fibrobacter sequences observed previously in clone libraries from freshwater environments. 16S rRNA gene sequences recovered in the same way from soil samples and ovine feces in the surrounding land were all F. succinogenes and did not include any from this group of the "freshwater" Fibrobacteres. In all cases, nested PCR was required to detect Fibrobacter 16S rRNA genes, and qPCR analysis of reverse transcribed bacterial community RNA confirmed their very low relative abundance on colonized cotton baits in the water column (at 0, 3, 7, 11, and 13 m) and on the sediment surface (<0.02% of total bacterial rRNA). However, in Esthwaite Water sediment itself, the relative abundance of fibrobacters was 2 orders of magnitude higher (ca. 1% of total bacterial rRNA). The presence of fibrobacters, including the cellulolytic rumen species F. succinogenes, on colonized cellulose samples and in lake sediment suggests that these organisms may contribute to the primary degradation of plant and algal biomass in freshwater lake ecosystems.

Histopathological and molecular evaluation of Holstein-Friesian cows postpartum: Toward an improved understanding of uterine innate immunity

Bovine uterine disease reduces milk yield, impairs fertility and has implications for animal welfare. During involution, the uterus is usually exposed to multiple potential bacterial pathogens which are cleared by successful orchestration of the local inflammatory response. Unsuccessful resolution leads to the development of disease. The aim of this study was to characterize the local innate immune response in the uterus during physiological involution using histopathological and molecular analyses in 9 cows, 2 weeks after calving (early postpartum, EPP), and 4 cows, 9 weeks after calving (late postpartum, LPP). Uterine biopsies taken from each cow were classified by histopathology, and RNA was extracted for molecular analysis. Two EPP cows were classified with a mild, 5 with a moderate and 2 with a severe inflammatory response. Relative gene expression analysis was then performed using quantitative real-time PCR (qRT-PCR) and specific primers for genes encoding Toll-like receptors (TLRs), chemokines, cytokines, acute phase proteins (APPs) and antimicrobial peptides (AMPs). TLR4, transcription factor NFKB1 and the inflammatory cytokines IFNG, IL1A, IL6, IL8, IL12A were all significantly increased in EPP cows ( P < 0.05). Increase in HP, SAA3, TAP and DEFB5 genes was particularly marked in cows with severe inflammation. These results reveal evidence of an inflammatory uterine environment in the early postpartum period with significant induction of both AMP and APP genes. Histopathological grades in EPP cows are underpinned by quantitative changes in gene expression. Understanding the molecular mechanisms contributing to uterine immunity in the early postpartum period may identify candidate genes associated with the resolution of inflammation.

Development of a quantitative real-time PCR assay for detection of Vibrio tubiashii targeting the metalloprotease gene

Vibrio tubiashii has recently re-emerged as a pathogen of bivalve larvae, causing a marked increase in the mortality of these species within shellfish rearing facilities. This has resulted in substantial losses of seed production and thus created the need for specific as well as sensitive detection methods for this pathogen. In this project, quantitative PCR (qPCR) primers were developed and optimized based upon analysis of the V. tubiashii vtpA gene sequence, encoding a metalloprotease known to cause larval mortality. Standard curves were developed utilizing dilutions of known quantities of V. tubiashii cells that were compared to colony forming unit (CFU) plate counts. The assay was optimized for detection of vtpA with both lab-grown V. tubiashii samples and filter-captured environmental seawater samples seeded with V. tubiashii. In addition, the primers were confirmed to specifically detect only V. tubiashii when tested against a variety of non-target Vibrio species. Validation of the assay was completed by analyzing samples obtained from a shellfish hatchery. The development of this rapid and sensitive assay for quantitative detection of V. tubiashii will accurately determine levels of this bacterium in a variety of seawater samples, providing a useful tool for oyster hatcheries and a method to assess the presence of this bacterium in the current turbulent ocean environment.